Stabilized nucleic acid dark quencher-fluorophore probes

ABSTRACT

The present invention provides a new class of solids supports for synthesis of modified oligomers of nucleic acids, and nucleic acid probes that have a format expediently synthesized on the new supports. Exemplary solid supports include at least one quencher bound through a linker to the solid support. Various exemplary embodiments include a moiety that stabilizes a duplex, triplex or higher order aggregation (e.g., hybridization) of nucleic acids of which the oligomer of the invention is a component. Other components of the solid support include moieties that stabilize aggregations of nucleic acids, e.g., intercalators, minor groove binding moieties, bases modified with a stabilizing moiety (e.g., alkynyl moieties, and fluoroalkyl moieties), and conformational stabilizing moieties, such as those described in commonly owned U.S. Patent Application Publication No. 2007/0059752.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a non-provisional filing claiming priority to U.S. ProvisionalPatent Application No. 61/041,515 filed Apr. 1, 2008, the disclosure ofwhich is incorporated herein by reference in its entirety for allpurposes.

TECHNICAL FIELD

The invention relates generally to novel materials (e.g., solid supportsand phosphoramidited) for nucleic acid synthesis. Exemplary materialsinclude a solid support and a phosphoramidite functionalized with astabilizing moiety. Various materials are functionalized with both astabilizing moiety and a quencher. The invention also provides nucleicacid monomers and oligomers, including fluorescent-labeled probessynthesized using these materials. Also provided are methods ofoligomer-based analyses and diagnosis utilizing oligomer probes of theinvention binding to nucleic acid target sequences.

BACKGROUND OF THE INVENTION

Fluorescent oligonucleotide probes are important tools for geneticanalysis, in both genomic research and development, and in clinicalmedicine. One particularly useful class of fluorescent probes is selfquenching probes, also known as fluorescence energy transfer probes, orFET probes. Although the design of different probes using this motif mayvary in detail, FET probes contain both a fluorophore and quenchertethered to an oligonucleotide. The fluorophore and the quencher areconfigured to produce a signal only as a result of hybridization to anintended target. Despite the limited availability of FET probes,techniques incorporating their use are rapidly displacing competitivemethods.

Probes containing a fluorophore-quencher pair have been developed forhybridization assays where the probe forms a hairpin structure, i.e.,where the probe hybridizes to itself to form a loop such that thequencher molecule is brought into proximity with the reporter moleculein the absence of a complementary nucleic acid sequence to prevent theformation of the hairpin structure (see, for example, WO 90/03446;European Patent Application No. 0 601 889 A2). When a complementarytarget sequence is present, hybridization of the probe to thecomplementary target sequence disrupts the hairpin structure and causesthe probe to adopt a conformation where the quencher molecule is nolonger close enough to the reporter molecule to quench the reportermolecule. As a result, the probes provide an increased fluorescentsignal when hybridized to a target sequence than when they areunhybridized. Probes including a hairpin structure can be difficult todesign and may interfere with the hybridization of the probe to thetarget sequence.

Assays have also been developed for identifying the presence of ahairpin structure using two separate probes, one containing a reportermolecule and the other a quencher molecule (see, Meringue, et al.,Nucleic Acids Research, 22: 920-928 (1994)). In these assays, thefluorescence signal of the reporter molecule decreases when hybridizedto the target sequence due to the quencher molecule being brought intoproximity with the reporter molecule.

One particularly important application for probes including areporter-quencher molecule pair is their use in nucleic acidamplification reactions, such as polymerase chain reactions (PCR), todetect the presence and amplification of a target nucleic acid sequence.In general, nucleic acid amplification techniques have opened broad newapproaches to genetic testing and DNA analysis (see, for example,Arnheim et al. Ann. Rev. Biochem., 61: 131-156 (1992)). PCR, inparticular, has become a research tool of major importance withapplications in, for example, cloning, analysis of genetic expression,DNA sequencing, genetic mapping and drug discovery (see, Arnheim et al.,supra; Gilliland et al., Proc. Natl. Acad. Sci. USA, 87: 2725-2729(1990); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992);Green et al, PCR Methods and Applications, 1: 77-90 (1991); Blackwell etal., Science, 250: 1104-1110 (1990)).

Commonly used methods for detecting nucleic acid amplification productsrequire that the amplified product be separated from unreacted primers.This is typically achieved either through the use of gelelectrophoresis, which separates the amplification product from theprimers on the basis of a size differential, or through theimmobilization of the product, allowing free primer to be washed away.However, three methods for monitoring the amplification process withoutprior separation of primer have been described. All of them are based onFRET, and none of them detect the amplified product directly. Instead,all three methods detect some event related to amplification. For thatreason, they are accompanied by problems of high background, and are notquantitative, as discussed below.

One method, described in Wang et al. (U.S. Pat. No. 5,348,853; Wang etal., Anal. Chem., 67: 1197-1203 (1995)), uses an energy transfer systemin which energy transfer occurs between two fluorophores on the probe.In this method, detection of the amplified molecule takes place in theamplification reaction vessel, without the need for a separation step.This method, however, does not detect the amplified product, but insteaddetects the dissociation of primer from the “energy-sink”oligonucleotide. Thus, this method is dependent on detection of adecrease in emissions; a significant portion of labeled primer must beutilized in order to achieve a reliable difference between the signalsbefore and after the reaction.

A second method detecting an amplification product without priorseparation of primer and product is the 5′-nuclease PCR assay (alsoreferred to as the TaqMan™ assay) (Holland et al., Proc. Natl. Acad.Sci. USA, 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res., 21:3761-3766 (1993)). This assay detects the accumulation of a specific PCRproduct by hybridization and cleavage of a doubly labeled fluorogenicprobe (the “TaqMan” probe) during the amplification reaction. Thefluorogenic probe consists of an oligonucleotide labeled with both afluorescent reporter dye and a quencher dye. During PCR, this probe iscleaved by the 5′-exonuclease activity of DNA polymerase if, and onlyif, it hybridizes to the segment being amplified. Cleavage of the probegenerates an increase in the fluorescence intensity of the reporter dye.

In the TaqMan assay, the donor and quencher are preferably located onthe 3′- and 5′-ends of the probe, because the requirement that 5′-3hydrolysis be performed between the fluorophore and quencher may be metonly when these two moieties are not too close to each other (Lyamichevet al., Science, 260:778-783 (1993). This requirement is a seriousdrawback of the assay as the efficiency of energy transfer decreaseswith the inverse sixth power of the distance between the reporter andquencher. Thus, if the quencher is not close enough to the reporter toachieve the most efficient quenching the background emissions fromunhybridized probe can be quite high.

Yet another method of detecting amplification products that relies onthe use of energy transfer is the “beacon probe” method described byTyagi et al. (Nature Biotech., 14:303-309 (1996)) which is also thesubject of U.S. Pat. Nos. 5,119,801 and 5,312,728 to Lizardi et al. Thismethod employs oligonucleotide hybridization probes that can formhairpin structures. On one end of the hybridization probe (either the5′- or 3′-end) there is a donor fluorophore, and on the other end, anacceptor moiety. In this method, the acceptor moiety is a quencher,absorbing energy from the donor. Thus when the beacon is in the openconformation, the fluorescence of the donor fluorophore is detectable,whereas when the beacon is in hairpin (closed) conformation, thefluorescence of the donor fluorophore is quenched. When employed in PCR,the molecular beacon probe, which hybridizes to one of the strands ofthe PCR product, is in “open conformation,” and fluorescence isdetected, while those that remain unhybridized will not fluoresce. As aresult, the amount of fluorescence will increase as the amount of PCRproduct increases, and thus can be used as a measure of the progress ofthe PCR.

Because this method is based on hybridization of the probe to a templateregion between the primer sequences, it has a number of problemsassociated with it. For example, it is unlikely that the beacon probeswill hybridize quantitatively to one strand of double-stranded PCRproduct, especially when the amplification product is much longer thanthe beacon probe.

Additional limitations have also impeded the application and use of FETprobes. First, currently available probe designs have a higherfluorescent noise background than is desirable. In some cases this isdue to the difficulty of purifying the probe which must be rigorouslypurged of any spurious fluorescent byproducts. As a result probes mustundergo at least 2 levels of purification before they are acceptable.This labor factor results in very high probe cost, approximately$300-$600 per probe. A second fundamental limitation is the inherentnoise of the probe itself which is a result of the physical geometry ofthe probe which places constraints on the fluorophore and quencherinteraction.

More recently, oligonucleotides have been shown to bind in asequence-specific manner to duplex DNA to form triplexes.Single-stranded nucleic acid, primarily RNA, is the target molecule foroligonucleotides that are used to inhibit gene expression by an“antisense” mechanism (Uhlmann, E., et al., Chem Reviews (1990)90:543-584; van der Krol, A. R., et al., Biotechniques (1988)6:958-976). Antisense oligonucleotides are postulated to exert an effecton target gene expression by hybridizing with a complementary RNAsequence. In this model, the hybrid RNA-oligonucleotide duplexinterferes with one or more aspects of RNA metabolism includingprocessing, translation and metabolic turnover. Chemically modifiedoligonucleotides have been used to enhance their nuclease stability.

Duplex DNA can be specifically recognized by oligomers based on arecognizable nucleomonomer sequence. Exemplary ecognition rules areoutlined by Maher III, L. J., et al., Science (1989) 245:725-730; Moser,H. E., et al., Science (1987) 238:645-650; Beal, P. A., et al., Science(1992) 251:1360-1363; Cooney, M., et al., Science (1988) 241:456-459;and Hogan, M. E., et al., EP Publication 375408.

Sequence-specific targeting of both single-stranded and duplex targetsequences has applications in diagnosis, analysis, and therapy. Undersome circumstances wherein such binding is to be effected, it isadvantageous to stabilize the resulting duplex or triplex over long timeperiods.

The use of triple helix (or triplex) complexes as a means for inhibitionof the expression of target gene expression was previously adduced(International Application No. PCT/US89/05769). Triple helix structureshave been shown to interfere with target gene expression (InternationalApplication No. PCT/US91/09321; Young, S. L. et al., Proc. Natl. Acad.Sci. (1991) 88:10023-10026), demonstrating the feasibility of thisapproach.

European Patent Application No. 92103712.3, Rahim, S. G., et al(Antiviral Chem. Chemother. (1992) 3:293-297), and InternationalApplication No. PCT/SE91/00653 describe pyrimidine nucleomonomers havingan unsaturated group in the 5-position. 5-Propynyl and 5-ethynyl groupsare among the described derivatives.

Synthesis of nucleomonomers having unsaturated alkyl groups at the5-position of uracil has been described (DeClercq, E., et al., J. Med.Chem. (1983) 26:661-666; Goodchild, J., et al., J. Med. Chem. (1983)26:1252-1257). Oligomers containing 5-propynyl modified pyrimidines havebeen described (Froehler, B. C., et al., Tetrahedron Letters (1992)33:5307-5310).

Conversion of 5-propynyl-2′-deoxyuridine, 5-butynyl-2′-deoxyuridine andrelated compounds to the 5′-triphosphate followed by incorporation ofthe monomer into oligomers by E. coli polymerase has been described(Valko, K., et al., J. Liquid Chromatog. (1989) 12:2103-2116; Valko, K.et al., J. Chromatog. (1990) 506:35-44). These studies were conducted asa structure to activity analysis of nucleotide analogs having a seriesof substitutions at the 5-position of uracil. The activity of thenucleotide analogs as substrates for E. coli polymerase was examined andcorrelated with characteristics such as the hydrophobicity of themonomer. No information was presented regarding the properties ofoligomers containing the analogs.

Oligomers having enhanced affinity for complementary target nucleic acidsequences would have improved properties for diagnostic applications,therapeutic applications and research reagents. Moreover, there existsin the art a need for improved probes for detecting nucleic acids (e.g.,amplification products) rapidly, sensitively, reliably andquantitatively. Ideal probes would give rise to minimal backgroundsignal and be easily and inexpensively prepared. Quite surprisingly, thepresent invention provides such probes. Oligomeric FET and FRET probesof the present invention have improved binding affinity for doublestranded and/or single stranded target sequences.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a scheme showing an exemplary preparation of a solid supportof the invention functionalized with a stabilizing moiety.

FIG. 2 is a scheme showing an exemplary preparation of a solid supportof the invention derivatized with functional moieties.

SUMMARY OF THE INVENTION

The present invention provides a new class of phosphoramidites and solidsupports for synthesis of modified nucleic acid oligomers, and nucleicacid probes (e.g., oligomers, e.g., oligonucleotides) of a formatexpediently synthesized using the phosphoramidites or on the newsupports. Exemplary solid supports include at least one quencher boundthrough a linker to the solid support. Various exemplary embodimentsprovide a solid support or a phosphoramidite functionalized a moietythat stabilizes a duplex, triplex or higher order aggregation (e.g.,hybridization) of the oligomer of the invention with a target nucleicacid. Exemplary components of the solid support (and the oligomers)include moieties that stabilize hybridization of nucleic acids, e.g.,intercalators, minor groove binding moieties, bases modified with astabilizing moiety (e.g., alkynyl moieties, and fluoroalkyl moieties),and conformational stabilizing moieties, such as those described incommonly owned U.S. Patent Application Publication No. 2007/0059752.Exemplary oligomers synthesized on the solid supports of the invention,or using the phosphoramidites of the invention include a quencher and astabilizing moiety. Various oligomers also include a fluorophore and,optionally, one or more additional detectable moiety, stabilizing orquencher moiety.

In an exemplary embodiment, the quencher linked to the solid support,phosphoramidite or oligomer of the invention is a member of a class ofquenchers in which a first substituted or unsubstituted aryl or firstsubstituted or unsubstituted heteroaryl moiety is linked to a secondsubstituted or unsubstituted aryl or second substituted or unsubstitutedheteroaryl moiety through an exocyclic diazo bond. In an exemplaryembodiment, the quenchers are essentially non-fluorescent (“darkquenchers”), and are optionally members of a class of compounds termed“Black Hole QuenchersTM” (“BHQs”), which are disclosed in commonly ownedU.S. Patent No. 7,109,312. The solid supports, phosphoramidites andoligomers functionalized with these quenchers can also be bound to oneor more conjugated components, generally covalently conjugated to thebase or sugar of the nucleic acid phosphoramidite, solid support oroligomer through a linker. An exemplary conjugated component is a minorgroove binder, an intercalator, a fluorocarbon hybridization stabilizingmoiety, an alkynyl hybridization stabilizing moiety (collectively,“stabilizing moieties”), a fluorophore and a quencher of fluorescentenergy.

In exemplary embodiments, the present invention provides solid supportsand phosphoramidites appropriate for synthesizing oligomeric nucleicacid probes including a stabilizing moiety, a quencher and/or afluorophore. Also provided are probes of a format expedientlymanufactured on such a solid support or using such a phosphoramidite.Exemplary oligomeric nucleic acid probes of the invention arecharacterized by interaction between a quencher and a fluorophore, eachconjugated to the oligomer in order to minimize the fluorescence of theprobe in the absence of its interaction with a target (e.g.,hybridization to a nucleic acid at least partially complementary to thetarget sequence).

In many dual-labeled nucleic acid probes, the interaction between thefluorophore and the quencher is brought about by using a nucleic acidprobe sequence that forms a secondary structure (e.g., hairpin, loop,etc.). Requiring that a probe adopt a secondary structure significantlycomplicates the design of the probe and greatly restricts the nucleicacid sequences that can be used as components of the probes. Incontrast, exemplary oligomeric nucleic acid probes of the inventionfacilitate the interaction between the quencher and the fluorophorewithout requiring concomitant formation of nucleic acid secondarystructure, thereby allowing a much greater diversity of nucleic acidsequences to be used as components of fluorescent probes. In variousembodiments, these probes include one or more Dark Quencher (e.g., BlackHole Quencher) as defined herein.

Moreover, by varying the number and identity of the members of theconjugated diazo-(hetero)aryl system of the quenchers used in thepresent invention the spectral properties (e.g., absorbance) of thequencher can be “tuned” to match the spectral characteristics (e.g.,emission) of one or more fluorophores. This characteristic providesoligomeric probes of the invention selectable to have a broad range ofabsorbance maxima. Accordingly the oligomeric probes of the inventionare well-suited for use in multiplexing applications. Furthermore, theinvention provides solid supports and probes useful in multiplexingapplications using one or more distinct fluorophore in combination withone or more quencher, thereby expanding the choices of donor-acceptorpairs that can be incorporated into the oligomeric probes. Accordingly,in various embodiments, the invention provides at least 2, at least 3,at or least 4 oligomeric probes each of which is functionalized with aquencher having a spectral property differentiatable from the sameproperty of the quencher in the other probes (e.g., emissionwavelength).

In an exemplary embodiment, the invention provides a compound having astructure according to Formula I:

wherein X^(a) is a stabilizing moiety selected from fluoroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. In various embodiments, X^(a) is a minor groove binder or anintercalating agent.

The symbols L¹, L², L³ and L⁴ represent linkers. The linkers areindependently selected from a single covalent bond, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkylmoieties. Y^(a) is a member selected from CR^(a), and N in which R^(a)is a member selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R^(a) is optionally a linkerto a functional component.

The symbol Z^(a) represents a solid support, OR^(b) or NR^(b)R^(b′) inwhich R^(b) and R^(b′) are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The symbol R^(c) represents a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and a phosphorus-containing linker covalentlybound to a nucleic acid. In an exemplary embodiment, R^(c) is a nucleicacid protecting group, e.g., dimethoxytrityl (“DMT”). In anotherembodiment, R^(c) is a phosphodiester linker to another nucleic acidmoiety, which is optionally derivatized with one or more functionalcomponent.

The compounds of the invention include a quencher of fluorescenceenergy. The quencher is represented by the symbol Q and, in exemplaryembodiments, includes one or more of the following structural features:

-   -   (a) at least three residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl, wherein the first residue is        covalently linked to the second residue via a first exocyclic        diazo bond. The first or the second residue is covalently linked        to a third residue through a second diazo bond; and    -   (b) at least two residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl. The first residue is covalently linked        to the second residue via an exocyclic diazo bond, and at least        one the residues is a member selected from substituted or        unsubstituted polycyclic aryl and substituted or unsubstituted        polycyclic heteroaryl groups.

The quenchers are linked to the remainder of the compound of theinvention via a linker. The quencher and the linker are coupled byreaction of a reactive functional group on a precursor quencher and areactive functional group on the linker. The two reactive functionalgroups are of complementary reactivity and upon reaction form a linkagefragment as defined herein.

Other objects, advantages and aspects of the present invention will beapparent from the detailed description below.

Detailed Description Of The Invention

Abbreviations

“BHQ,” as used herein, refers generally to dark quenchers including oneor more diazo bond and specifically to “Black Hole QuenchersTM.”Exemplary BHQ's of use in the present invention are described in U.S.Patent No. 7,109,312. “FET,” as used herein, refers to “FluorescenceEnergy Transfer.” “FRET,” as used herein, refers to “FluorescenceResonance Energy Transfer.” These terms are used herein to refer to bothradiative and non-radiative energy transfer processes. For example,processes in which a photon is emitted and those involving long rangeelectron transfer are included within these terms. Throughout thisspecification, both of these phenomena are subsumed under the generalterm “donor-acceptor energy transfer.” “SNP” refers to “SingleNucleotide Polymorphism.”

Definitions

The following definitions are broadly applicable to each of theembodiments of the present invention set forth hereinbelow. Unlessdefined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Molecular biological techniques andprocedures are generally performed according to conventional methods inthe art and various general references (see generally, Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference). The nomenclature used herein and the laboratoryprocedures in analytical chemistry, and organic synthesis are those wellknown and commonly employed in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight- or branched-chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di-, tri- andtetra-valent radicals, having the number of carbon atoms designated(i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, also optionally include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”. The term “alkyl”, as used herein refers to alkyl,alkenyl and alkynyl moieties, each of which can be mono-, di- orpolyvalent species as appropriate to satisfy valence requirements. Alkylgroups are optionally substituted, e.g., with one or more groupsreferred to herein as an “alkyl group substituent.”

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl moiety, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. For alkylene andheteroalkylene linker groups, it is optional that no orientation of thelinker group is implied by the direction in which the formula of thelinker group is written. For example, the formula —C(O)₂R′—represents—C(O)₂R′— and, optionally, —R′C(O)₂—. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight, seven, six, five or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight- or branched-chain, orcyclic alkyl radical consisting of the stated number of carbon atoms andat least one heteroatom selected from the group consisting of B, O, N,Si and S, wherein the heteroatom may optionally be oxidized and thenitrogen atom may optionally be quaternized. The heteroatom(s) may beplaced at any internal position of the heteroalkyl group or at aterminus of the chain, e.g., the position through which the alkyl groupis attached to the remainder of the molecule. Examples of “heteroalkyl”groups include, but are not limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Two or more heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent refers to asubstituted or unsubstituted divalent heteroalkyl radical, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like).

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings, one or more of which is optionally acycloalkyl or heterocycloalkyl), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group of “arylgroup substituents” described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) optionally includes both homoaryland heteroaryl rings as defined above. Thus, the term “arylalkyl”optionally includes those radicals in which an aryl group is attached toan alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like)including those alkyl groups in which a carbon atom (e.g., a methylenegroup) has been replaced by, for example, an oxygen atom (e.g.,phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and thelike).

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” includes groups with carbon atoms bound to groups otherthan hydrogen, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl(e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like). Exemplary alkylgroup substituents include those groups referred to herein as “reactivefunctional groups” and “linkage fragments.” In various embodiments, thealkyl group substituent is a phosphorus-containing moiety, e.g., aphosphodiester or a phosphodiester modification such as those describedherein.

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” Exemplary substituents are selectedfrom the list of alkyl group substituents and others, for example:halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″ and R″″ are preferably independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl andsubstituted or unsubstituted heteroaryl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer from 0 to 3. Alternatively,two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—, —NR—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 4. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₁₆)alkyl. Exemplaryaryl group substituents include those groups referred to herein as“reactive functional groups” and “linkage fragments.”

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S) and silicon (Si).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups. R can also refer to alkyl groupsubstituents and aryl group substituents.

The term “salt(s)” includes salts of the compounds which are preparedwith relatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When compounds ofthe present invention contain relatively acidic functionalities, baseaddition salts can be obtained by contacting the neutral form of suchcompounds with a sufficient amount of the desired base, either neat orin a suitable inert solvent. Examples of base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of acid addition salts include those derived from inorganicacids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids, and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,butyric, maleic, malic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate, and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge et al., Journal of Pharmaceutical Science, 66: 1-19 (1977)).Certain specific compounds of the present invention contain both basicand acidic functionalities that allow the compounds to be converted intoeither base or acid addition salts. Hydrates of the salts are alsoincluded.

As used herein, “nucleic acid” means nucleosides, nucleotides andoligonucleotides, e.g., DNA, RNA, whether single-stranded,double-stranded, or in more highly aggregated hybridization motifs, andany chemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,peptide nucleic acids (PNAs), phosphodiester group modifications (e.g.,phosphorothioates, methylphosphonates), sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat exocyclic amines, substitution of 4-thiouridine, substitution of5-bromo or 5-iodo-uracil; backbone modifications, methylations (e.g., 5′and/or 3′), unusual base-pairing combinations such as the isobases,isocytidine and isoguanidine and the like. Nucleic acids can alsoinclude non-natural bases. A “nucleomonomer” refers to a single nucleicacid unit, which can be a nucleoside, nucleotide or a modificationthereof.

“Base” as used herein includes those moieties which contain not only theknown purine and pyrimidine heterocycles and the invention pyrimidines,but also heterocycle analogs and tautomers thereof. Purines includeadenine, guanine and xanthine and exemplary purine analogs include8-oxo-N⁶-methyladenine and 7-deazaxanthine. Pyrimidines include uraciland cytosine and their analogs such as 5-methylcytosine, 5-methyluraciland 4,4-ethanocytosine. This term also encompasses non-natural bases.Representative non-natural bases include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,nitroindole, and 2,6-diaminopurine.

In various embodiments, the inventive compounds include pyrimidinesderivatized at the 5-position. The derivatives are 1-alkenyl-,1-alkynyl-, heteroaromatic- and 1-alkynyl-heteroaromatic modifications.“1-Alkenyl” means an olefinically-unsaturated (double bond containing)acyclic group. “1-Alkynyl” means an acetylenically-unsaturated (triplebond containing) acylic group

As used herein, “nucleoside” means a subset of nucleic acid in which abase is covalently attached to a sugar or sugar analog and whichoptionally includes a phosphite, phosphoramidite or phosphine. The termnucleoside includes ribonucleosides, deoxyribonucleosides, or any othernucleoside which is an N-glycoside or C-glycoside of a base. Thestereochemistry of the sugar carbons can be other than that of D-ribose.Nucleosides also include those species which contain modifications ofthe sugar moiety, for example, wherein one or more of the hydroxylgroups are replaced with a halogen, a heteroatom, an aliphatic groups,or are functionalized as ethers, amines, thiols, and the like. Thepentose moiety can be replaced by a hexose or an alternate structuresuch as a cyclopentane ring, a 6-member morpholino ring and the like.Nucleosides as defined herein also include a base linked to an aminoacid and/or an amino acid analog having a free carboxyl group and/or afree amino group and/or protected forms thereof. Nucleosides alsooptionally include one or more base modification, e.g., modified with afluorocarbyl, alkenyl or alkynyl moiety. A nucleoside including aphosphodiester or phosphodiester modification, is referred to herein asa nucleotide.

“Sugar modification,” as used herein, means any pentose or hexose moietyother than 2′-deoxyribose. Modified sugars include, for example,D-ribose, 2′-O-alkyl, 2′-amino, 2′-halo functionalized pentoses, hexosesand the like. Exemplary sugar modifications include those sugars inwhich one or more of the hydroxyl groups is replaced with a halogen, aheteroatom, an alkyl moiety, or are functionalized as ethers, esters,and the like. The pentose moiety can be replaced by a hexose or analternate structure such as a cyclopentane ring, a 6-member morpholinoring and the like. Nucleosides as defined herein are also intended toinclude a base linked to an amino acid and/or an amino acid analoghaving a free carboxyl group and/or a free amino group and/or protectedforms thereof. Sugars having a stereochemistry other than that of aD-ribose are also included.

“Phosphodiester group modification” means any analog of the nativephosphodiester group that covalently couples adjacent nucleomonomers.Substitute linkages include phosphodiester analogs, e.g. such asphosphorothioate and methylphosphonate, and nonphosphorus containinglinkages, e.g. such as acetals and amides.

Nucleic acid modification also include 3′ and 5′ modifications such ascapping with a quencher (e.g., a BHQ), a fluorophore, intercalator,minor groove binder, a fluorocarbon, a conformationally assistedstabilizing group or another moiety. In various embodiments, the cappinggroup is covalently conjugated to the oligomer through a linker group.

Oligomers are defined herein as two or more nucleomonomers covalentlycoupled to each other by a phosphodiesester or modified phosphodiestermoiety. Thus, an oligomer can have as few as two nucleomonomers (adimer), and have essentially no upper limit of nucleomonomers. Oligomerscan be binding competent and, thus, can base pair with cognatesingle-stranded or double-stranded (or higher order aggregation) nucleicacid sequences. Oligomers are also useful as synthons for longeroligomers as described herein. Oligomers can also contain abasic sitesand pseudonucleosides. In various embodiments, the oligomers of theinvention are functionalized. The moieties functionalizing the oligomersare discussed below. In describing certain embodiments the term“oligomer” is used interchangeably to refer to the nucleic acid sequenceof the oligomer, the modified nucleic acid sequence providing a probe orthe modified nucleic acid sequence providint a solid support of theinvention “Peptide” refers to an oligomer in which the monomers areamino acids and are joined together through amide bonds, alternativelyreferred to as a polypeptide. When the amino acids are α-amino acids,either the L-optical isomer or the D-optical isomer can be used.Additionally, unnatural amino acids, for example, β-alanine,phenylglycine and homoarginine are also included. Commonly encounteredamino acids that are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-isomer. The L-isomers are generally preferred. Inaddition, other peptidomimetics are also useful in the presentinvention. For a general review, see, Spatola, A. F., in CHEMISTRY ANDBIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983).

A “solid support” is a solid material having a surface for attachment ofmolecules, compounds, cells, or other entities, or to which surface suchspecies are attached. The surface of a solid support can be flat orotherwise configured. A solid support can be porous or non-porous. Asolid support can be a chip or array that comprises a surface, and thatmay comprise glass, silicon, nylon, polymers, plastics, ceramics, ormetals. A solid support can also be a membrane, such as a nylon,nitrocellulose, or polymeric membrane, or a plate or dish and can becomprised of glass, ceramics, metals, or plastics, such as, for example,a 96-well plate made of, for example, polystyrene, polypropylene,polycarbonate, or polyallomer. A solid support can also be a bead orparticle of any shape, and is preferably spherical or nearly spherical,and preferably a bead or particle has a diameter or maximum width of 1millimeter or less, more preferably of between 0.1 to 100 microns. Suchparticles or beads can be comprised of any suitable material, e.g.,glass or ceramics, and/or one or more polymers, such as, for example,nylon, polytetrafluoroethylene, TEFLON™, polystyrene, polyacrylamide,sepaharose, agarose, cellulose, cellulose derivatives, or dextran,and/or can comprise metals, particularly paramagnetic metals, such asiron.

Supports for solid phase synthesis are known in the art and include, butare not limited to, high cross-linker polystyrene (McCollum, et al.,Tetrahedron Lett. 32: 4069-4072 (1991), polystyrene/PEG copolymer (Gao,et al., Tetrahedron Lett. 32: 5477-5480 (1991), silica gel (Chow, etal., Nucl. Acids Res. 9: 2807-2817 (1981)), polyamide bonded silica gel(Gait, et al., Nucl. Acids Res. 10: 6243-6254 (1982)), cellulose (Crea,et al., Nucl. Acids Res. 8: 2331-2348 (1980)), (and controlled poreglass (CPG) (Koster, et al., Tetrahedron Lett. 24: 747-750 (1983). Anexemplary solid support is CPG beads. CPG beads can be derivatized forthe attachment of a nucleomonor or oligomer in a variety of ways. Forexample, CPG beads can be treated with 3-aminopropyltriethoxysilane toadd an amino propyl linker handle for the attachment of oligonucleotideanalogue monomers or dimers (Koster, et al., Tetrahedron Lett. 24:747-750 (1983), or, preferably, a long-chain alkylamine group, mostpreferably including a terminal nucleoside, can be attached to CPG(Adams, et al., J. Am. Chem. Soc. 105: 661-663 (1983)). Supports foroligonucleotide synthesis or peptide synthesis, for example dT-LCAA-CPG(Applied Biosystems), are commercially available.

An “intercalator” refers to a planar aromatic or heteroaromatic moietythat is capable of partial insertion and stacking between adjacentnucleobases. These moieties may be small molecules or part of a largerentity, such as a protein. Non-limiting examples of intercalatorsinclude acridines, anthracenes, anthracyclines, anthracyclinone,methylene blue, indole, anthraquinone, quinoline, isoquinoline,dihydroquinones, tetracyclines, psoralens, coumarins, ethidium halides,ethidium homodimers, homodimeric oxazole yellow (YOYO), thiazole orange(TOTO), dynemicins, 1,10-phenanthroline-copper, calcheamicin,porphyrins, distamycins, netropcins, and viologens.

A “minor groove binder” refers to a moiety typically having a molecularweight of approximately 150 to approximately 2000 Daltons. The moietybinds in a non-intercalating manner into the minor groove of doublestranded (or higher order aggregation) DNA, RNA or hybrids thereof,preferably, with an association constant greater than approximately 10³M⁻¹. Minor groove binding compounds have widely varying chemicalstructures, however, exemplary minor groove binders have a crescentshape three dimensional structure. Exemplar include certain naturallyoccurring compounds such as netropsin, distamycin and lexitropsin,mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, aswell as further related antibiotics and synthetic derivatives. Certainbisquarternary ammonium heterocyclic compounds, diarylamidines such aspentamidine, stilbamidine and berenil, CC-1065 and related pyrroloindoleand indole polypeptides, Hoechst 33258, 4′-6-diamidino-2-phenylindole(DAPI) as well as a number of oligopeptides consisting of naturallyoccurring or synthetic amino acids are minor groove binder compounds.Exemplary minor groove binders are described in U.S. Pat. No. 6,084,102.This type of binding can be detected by well establishedspectrophotometric methods, such as ultraviolet (u.v.) and nuclearmagnetic resonance (nmr) spectroscopy and also by gel electrophoresis.Shifts in u.v. spectra upon binding of a minor groove binder molecule,and nmr spectroscopy utilizing the “Nuclear Overhauser” (NOSEY) effectare particularly well known and useful techniques for this purpose. Gelelectrophoresis detects binding of a minor groove binder to doublestranded DNA or fragment thereof, because upon such binding the mobilityof the double stranded DNA changes.

The minor groove binder is typically attached to the oligomer or solidsupport through a linker comprising a chain about 20, about 15 atoms,about 10 or about 5 atoms.

Intercalating moieties or agents are readily distinguished from minorgroove binders on the basis that the intercalating agents are flataromatic (preferably polycyclic) molecules versus the “crescent shape”or analogous geometry of the minor groove binders. An experimentaldistinction can also be made by nmr spectroscopy utilizing the NuclearOverhauser effect.

The term “linker” or “L”, as used herein, refers to a single covalentbond (“zero-order”) or a series of stable covalent bonds incorporating1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S,Si and P that covalently link together the components of the compoundsof the invention, e.g., linking a solid support to a stabilizing agent,a quencher, a nucleomonor or oligomer of the invention; or linking aquencher or stabilizing moiety to a base in an amidite of the invention.Exemplary linkers include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30non-hydrogen atoms. Unless otherwise specified, “linking,” “linked,”“linkage,” “conjugating,” “conjugated” and analogous terms relating toattachment refer to techniques utilizing and species incorporatinglinkers. Exemplary linkers include a linkage fragment as defined herein.Moreover, a linker is of use to attach an oligomer or nascent oligomer(during oligomer synthesis) to the solid support of the invention. Thus,the invention also provides an oligomer of the invention covalentlyattached to a solid support (e.g., a solid support of the invention)through a linker. The solid supports and oligomers of the inventionoptionally include a cleavable linker between two components of thesolid support and oligomer (e.g., between the oligomer and the solidsupport, between the fluorophore and oligomer, between the quencher andoligomer, between the fluorophore and quencher, etc.). In variousembodiments, the linker joining the solid support to the oligomer is acleavable linker.

A “cleavable linker” is a linker that has one or more cleavable groupsthat may be broken by the result of a reaction or condition. Anexemplary cleavable linker is located within L² of Formula I, serving toallow for the expedient separation of a synthesized oligomer of theinvention from the solid support upon which it was synthesized. The term“cleavable group” refers to a moiety that allows for release of acomponent of the solid support or oligomer of the invention by cleavinga bond linker the released moiety to the remainder of the conjugate.Exemplary cleavage mechanisms of use both in preparing and using theoligomers and solid supports of the invention are enzymatically orotherwise chemically mediated.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamateesters, benzoin esters), and heat. Many cleaveable groups are known inthe art. See, for example, Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990);Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur.J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989).Moreover a broad range of cleavable, bifunctional (both homo- andhetero-bifunctional) spacer arms are commercially available.

An exemplary cleavable group is cleavable by a reagent, e.g. sodiumhydroxide, ammonia or other amine. In various embodiments the cleavablelinker is readily cleaved at room temperature or under microwaveconditions. In one embodiment, the cleavable linker is L² and it iscleaved by treatment with an amine, e.g., ammonia or an essentiallyanhydrous amine in an organic solvent.

A “linkage fragment,” is a moiety that links two or more components(e.g., functional component, solid support or linker). This term refersto a covalent bond that is formed by reaction of complementary reactionpartners, each of which has a reactive functional group of reactivitycomplementary to the reactivity of its partner. Linkage fragments in thesolid support and oligomers of the invention are independently selected.Exemplary linkage fragments include, but are not limited to S, SC(O)NH,HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O, and OC(O)NH, CH₂S,CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O, (CH₂)_(o)S or (CH₂)_(o)Y^(x)-PEGwherein, Y^(x) is S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH, or O and o isan integer from 1 to 50. In each of these exemplary linkage fragments,NH can be NR^(t). in which R^(t) is substituted or unsubstituted alkylor substituted or unsubstituted heteroalkyl. A linkage fragment can alsobe a phosphodiester or phosphodiester modification. In variousembodiments, the linkage fragment is between a linker and a fluorophore,a linker and a quencher, a linker and a stabilizing moiety or a linkerand a solid support. In an exemplary embodiment of the solid support andoligomers of the invention, each linkage fragment is a different linkagefragment.

The term “fluorophore” as used herein refers to a moiety that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, or metabolism by anenzyme, i.e., fluorogenic. Fluorophores may be substituted to alter thesolubility, spectral properties or physical properties of thefluorophore. Numerous fluorophores are known to those skilled in the artand include, but are not limited to coumarins, acridines, furans,dansyls, cyanines, pyrenes, naphthalenes, benzofurans, quinolines,quinazolinones, indoles, benzazoles, borapolyazaindacenes, oxazines andxanthenes, with the latter including fluoresceins, rhodamines, rosaminesand rhodols. These and other fluorophores of use in the invention aredescribed in Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBESAND RESEARCH CHEMICALS. Further useful fluorophores are described incommonly owned U.S. Patent Application Publication No. 2005/0214833 and2005/0170363 and herein below.

As used herein, “quencher” refers to any fluorescence-modifying moietyof the invention that can attenuate at least partly the light emitted bya fluorophore. This attenuation is referred to herein as “quenching”.Hence, in various embodiments, excitation of the fluorophore in thepresence of the quenching group leads to an emission signal that is lessintense than expected, or even completely absent. Quenching typicallyoccurs through energy transfer between the excited fluorophore and thequenching group.

The fluorophore or quencher may include substituents enhancing adesirable property, e.g., solubility in water, cell permeability andspectral absorption and emission, relative to the “parent” compound inthe absence of such substituent. As such the fluorophore or quencher ofuse in the invention include substituents that enhance a desirableproperty relative to an identical parent compound in the absence of theimproving substituent.

A “functional component” is a generic term for a moiety in a compound ofthe invention having a structure selected from a quencher, a fluorophoreand a stabilizing moiety (including, but not limited to, intercalators,minor groove binding moieties, bases modified with a stabilizing moiety(e.g., alkynyl moieties, and fluoroalkyl moieties), and conformationalstabilizing moieties, such as those described in commonly owned U.S.Patent Application Publication No. 2007/0059752).

The expression “amplification of polynucleotides” includes but is notlimited to methods such as polymerase chain reaction (PCR), ligationamplification (or ligase chain reaction, LCR) and amplification methodsbased on the use of Q-beta replicase. These methods are well known andwidely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a (forLCR). Reagents and hardware for conducting PCR are commerciallyavailable. Primers useful to amplify sequences from a particular generegion are preferably complementary to, and hybridize specifically tosequences in the target region or in its flanking regions. Nucleic acidsequences generated by amplification may be sequenced directly.Alternatively the amplified sequence(s) may be cloned prior to sequenceanalysis. A method for the direct cloning and sequence analysis ofenzymatically amplified genomic segments has been described by Scharf(1986). The present invention provides oligomeric primers of use inamplification processes. Moreover, there is provided a solid support ofuse in synthesizing such primers. In addition to primers, the inventionprovides probes, and methods of using such probes, to detect,characterize and/or quantify the products of amplification: alsoprovided are solid supports of use to synthesize these oligomericprobes.

The term “base-stacking perturbations” refers to any event that causes aperturbation in base-stacking such as, for example, a base-pairmismatch, a protein binding to its recognition site, or any otherentities that form oligonucleotide adducts. Various probes of theinvention are capable of detecting, characterizing and/or quantifyingsuch base-stacking perturbations. Moreover, the invention provides solidsupports of use in synthesizing probes capable of detecting,characterizing and/or quantifying such base-stacking perturbations.

The term “hybridized” refers to two nucleic acid strands associated witheach other which may or may not be fully base-paired: generally, thisterm refers to an association including an oligomer of the inventionwhether bound to a solid support or in solution.

The term “denaturing” refers to the process by which strands of nucleicacid duplexes (or higher aggregation) are no longer base-paired byhydrogen bonding and are separated into single-stranded molecules.Methods of denaturation are well known to those skilled in the art andinclude thermal denaturation and alkaline denaturation. This termgenerally refers to the dissociation of a probe of the invention fromits target nucleic acid.

The term “mismatches” refers to nucleic acid bases within hybridizednucleic acid duplexes (or higher aggregation) which are not 100%complementary. A mismatch includes any incorrect pairing between thebases of two bases located on complementary strands of nucleic acid thatare not the Watson-Crick base-pairs, e.g., A:T or G:C. The lack of totalhomology may be due to deletions, insertions, inversions, substitutionsor frameshift mutations. In various embodiments, the oligomer of theinvention includes a mismatch relative to its target nucleic acid,preferably allowing detection and/or characterization and/orquantification of the corresponding mismatch in its target. In certainembodiments, the mismatch is a single nucleotide mismatch.

As used herein, the term “polymorphism” refers to a sequence variationin a gene, and “mutation” refers to a sequence variation in a gene thatis associated or believed to be associated with a phenotype. The term“gene” refers to a segment of the genome coding for a functional productprotein control region. Polymorphic markers used in accordance with thepresent invention for subject identification may be located in coding ornon-coding regions of the genome, and various probes of the inventionare designed to hybridize to nucleic acid regions including thesemarkers. The term “subject,” as used herein refers to a subjectproviding a test sample from which target nucleic acids are obtained forthe purpose of genetic testing. The oligomers of the invention are ofuse in detecting and/or characterizing and/or quantifying polymorphismsand mutations. Moreover, the solid supports of the invention are of usein synthesizing oligomers of use to detect and/or characterize and/orquantitate polymorphisms and mutations.

The term “probe” as used herein refers to nucleic acid oligomersprepared using a solid support or amidite of the invention. In variousembodiments, the probes produce a detectable response upon interactionwith a binding partner. The probes include at least one detectablemoiety, or a pair of moieties that form an energy transfer pairdetectable upon some change of state of the probe in response to itsinteraction with a binding partner. The present invention providesprobes and amidites and solid supports of use to synthesize probes.Exemplary probes of the invention are of use to detect a polymorphism.In various embodiments, the polymorphism is a single nucleic acidpolymorphism (SNP).

The term “detectable response” as used herein refers to a change in oran occurrence of, a signal that is directly or indirectly detectableeither by observation or by instrumentation and the presence of or,preferably, the magnitude of which is a function of the presence of atarget binding partner for a probe in the test sample. Typically, thedetectable response is an optical response from a fluorophore resultingin a change in the wavelength distribution patterns or intensity ofabsorbance or fluorescence or a change in light scatter, fluorescencequantum yield, fluorescence lifetime, fluorescence polarization, a shiftin excitation or emission wavelength or a combination of the aboveparameters. The detectable change in a given spectral property isgenerally an increase or a decrease. However, spectral changes thatresult in an enhancement of fluorescence intensity and/or a shift in thewavelength of fluorescence emission or excitation are also useful. Thechange in fluorescence on ion binding is usually due to conformationalor electronic changes in the indicator that may occur in either theexcited or ground state of the fluorophore, due to changes in electrondensity at the ion binding site, due to quenching of fluorescence by thebound target metal ion, or due to any combination of these or othereffects. Alternatively, the detectable response is an occurrence of asignal wherein the fluorophore is inherently fluorescent and does notproduce a change in signal upon binding to a metal ion or biologicalcompound. The present invention provides probes providing a detectableresponse and solid supports of use to synthesize such probes.

Introduction

The present invention provides phosphoramidites, solid supports andoligomers of a format readily prepared on these solid supports.Exemplary oligomers are stabilized with respect to their ability tohybridize to target nucleic acid sequences, forming duplexes ortriplexes. Various oligomers of the invention are suitable for bindingto DNA duplex target sequences via either CT or GT triple helix bindingmotif.

In various embodiments, the oligomers of the invention are resistant tonuclease degradation relative to an oligodeoxynucleotide having nomodifications. Nuclease resistant oligomers of the invention areadvantageously used under conditions where nucleases are present. Forcertain applications, such as modulation of gene expression by via anantisense mechanism, nuclease stability by oligomers of the invention isan important functional aspect of the oligomer.

An additional aspect of the invention includes methods of detecting thepresence, absence or amount of a particular single-stranded DNA or RNAor a particular target duplex in a biological (or other) sample usingthe oligomers of the invention, to detect selected nucleic acidsequences. Such sequences can be associated with the presence ofneoplastic growth, viruses or disease conditions.

Exemplary oligomers of the invention have enhanced binding propertieswith respect to complementary single-stranded and double-strandednucleic acid sequences as compared to unmodified oligomers not havingthe stabilizing moiety component of the oligomers of the invention. Invarious embodiments, triple helix structures can be formed atphysiological pH levels of 7.0 and higher. Improved duplex formation isalso provided, in exemplary embodiments.

The invention provides oligomers useful for, in exemplary embodiments,(1) modulating gene expression in cells in vitro including cells grownin tissue culture (e.g., to treat a condition, e.g., cancer, infection,etc.), and (2) detecting and/or quantitating target nucleic acidsequences.

The invention is also directed to reagents and kits comprising thephosphoramidites, solid supports and/or oligomers of the invention.

The Embodiments

In an exemplary embodiment, the invention provides a compound having astructure according to Formula I:

wherein X^(a) is a stabilizing moiety selected from fluoroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. In various embodiments, X^(a) is a minor groove binder or anintercalating agent.

The symbols L¹, L², L³ and L⁴ represent linkers. The linkers areindependently selected from a single covalent bond, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkylmoieties. Y^(a) is a member selected from CR^(a), and N in which R^(a)is a member selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. R^(a) is optionally a linkerto a functional component.

The symbol Z^(a) represents a solid support, OR^(b) or NR^(b)R^(b′) inwhich R^(b) and R^(b′) are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The symbol R^(c) represents a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and a phosphorus-containing linker covalentlybound to a nucleic acid. In an exemplary embodiment, R^(c) is a nucleicacid protecting group, e.g., dimethoxytrityl (“DMT”). In anotherembodiment, R^(c) is a phosphodiester linker to another nucleic acidmoiety, which is optionally derivatized with one or more functionalcomponent.

The compounds of the invention include a quencher of fluorescenceenergy. The quencher is represented by the symbol Q and, in exemplaryembodiments, includes one or more of the following structural features:

-   -   (a) at least three residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl, wherein the first residue is        covalently linked to the second residue via a first exocyclic        diazo bond. The first or the second residue is covalently linked        to a third residue through a second diazo bond; and    -   (b) at least two residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl. The first residue is covalently linked        to the second residue via an exocyclic diazo bond, and at least        one the residues is a member selected from substituted or        unsubstituted polycyclic aryl and substituted or unsubstituted        polycyclic heteroaryl groups.

The quenchers are linked to the remainder of the compound of theinvention via a linker. The quencher and the linker are coupled byreaction of a reactive functional group on a precursor quencher and areactive functional group on the linker. The two reactive functionalgroups are of complementary reactivity and upon reaction form a linkagefragment as defined herein.

Exemplary linkers in Formula I include:

For L¹,

for example

in which z is an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

For L²,

for example

in which y, w and u are independently selected integers from 1 to 20,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20.

For L³,

for example

in which v, t and s are independently selected integers from 1 to 20,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20. The symbol X^(b) represents a member selected from O and S.X^(c) is a member selected from OR⁸, SR⁸ and NR⁸ R^(8a), in which R⁸ andR^(8a) are members independently selected from H, and substituted orunsubstituted alkyl, or the compound is a salt and OR⁸ and SR⁸ areselected from O⁻M⁺ and S⁻M⁺. M+ is any cation capable of forming a saltwith the negatively charged ion, including a metal ion or an ammoniumion. R⁹ is a member selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl and substituted or unsubstituted heteroaryl and a nucleic acidoptionally connected through a phosphorus-containing linker.

For L¹-Y^(a)-L³

for example

in which the indeces and variable radicals are as set forth above.

For L¹-Y^(a)-L²

for example

in which the indeces and variable radicals are as set forth above.For L¹-Y^(a)-L³,

for example

in which the indeces and variable radicals are as set forth above.

In exemplary embodiments, the invention provides a compound having astructure according to Formula VII:

in which the indices and variable radicals are as set forth above.

In selected embodiments, the invention includes a compound having astructure according to Formula VIII:

in which Q¹ is a fragment of the quencher. The fragment comprises amember selected from:

-   -   (a) two moieties selected from substituted or unsubstituted aryl        and substituted or unsubstituted heteroaryl, said two moieties        being linked through an exocyclic diazo bond; and    -   (b) a moiety selected from substituted or unsubstituted        polycyclic aryl and substituted or unsubstituted polycyclic        heteroaryl groups.        Each R¹² is a member independently selected from the group of        aryl substituents as defined herein; and the integer p is 0, 1,        2, 3, or 4. The remaining indeces and radicals are as set forth        above.

With respect to linkers of use in the compounds of the presentinvention, an exemplary embodiment is provided in which Z^(a) is a solidsupport and L²-Z^(a) comprises a structure according to Formula IX:

in which the indeces are as set forth above; and SS is the solidsupport.

In certain embodiments of the invention there is included on the solidsupport or a phosphoramidite or on an oligomer synthesized on the solidsupport a stabilizing moiety. An exemplary compound of the inventionaccording to any of the above-described embodiments is one in whichX^(a) is selected from intercalating agents and minor groove binders.

In various embodiments, the solid support or phosphoramidite or oligomerof the invention includes a base having a formula selected from:

in which R¹⁰ is a member selected from an alkynyl and a fluoroalkylmoiety.

By “alkynyl” is meant an acetylenically-unsaturated acylic group, suchas ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, 1,3-pentadiynyl,phenylethynyl, phenylethynyl, pyridine-ethynyl, pyrimidine-ethynyl,triazine-ethynyl, thiophene-ethynyl, thiazole-ethynyl andimidazole-ethynyl. Exemplary substituted groups include substituted orunsubstituted C₁-C₁₀ alkynyl groups, e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇,C₈, C₉, or C₁₀ alkynyl. substituted with 2-, 3-, and 4-pyridinyl (e.g.,2-, 3- and 4-pyrimidine-ethynyl), triazine (e.g., triazinyl-ethynyl),2-, 4- and 5-pyrimidinyl, 2-, 4- and 5-thiazolyl, 1-methyl-2-imidazolyl,2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 3-pyridinyl, 4-pyridinyl,2-pyridinyl, 2- and 3-furanyl-ethynyl, 2- and 3-thienyl-ethynyl, 2- and4-imidazolyl-ethynyl, 2-, 4- and 5-thiazoyl-ethynyl, 2-, 4- and5-oxazolyl-ethynyl, 2- and 3-pyrrolyl-ethynyl, 2- and 3-thienyl, 2- and3-furanyl, 2- and 3-pyrrolyl, propenyl (—CH≡CH—CH₃), vinyl and —C≡C-Z′where Z′ is hydrogen (H) or C₁-C₁₀ alkyl, haloalkyl (C₁-C₁₀ with 1 to 6halogen atoms or heteroalkyl (C₁-C₁₀ with 1 to 3 heteroatoms selectedfrom the group consisting of O, N and S). The alkynyl moiety can also bethe component of a linker or a linkage fragment.

Exemplary fluoroalkyl groups include linear (e.g., C₁-C₂₀, C₂-C₁₆,C₃-C₁₀, C₄-C₈) and cycloalkyl (e.g., C₃-C₈) moieties substituted withone or more fluoro moiety. Exemplary fluoroalkyl groups include 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. Perfluoro compounds are of use in the compounds of the invention.

Monomers

In various embodiments, the invention provides monomeric nucleic acidsof use in synthesizing oligormers. In a representative embodiment, themonomeric nucleic acid is a phosphoramidite bearing a stabilizingmoiety. An exemplary monomeric nucleic acid according to this embodimenthas the formula:

In various embodiments, the stabilizing moiety is an alkyne residue,providing a monomeric nucleic acid having the formula:

in which Q is a quencher, and L⁴ is a linker. Exemplary linkers includezero-order and substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl. R^(d) is H, substituted or unsubstitutedalkyl, or substituted or unsubstituted heteroalkyl. In an exemplaryembodiment, R^(d) is a nucleic acid protecting group, e.g., DMT. R^(c)is H or is a component of a phosphoramidite, e.g., —OPN(i-Pr)₂(OCNE).The ring labeled B represents a base as defined herein. Exemplary basesinclude:

in which R¹⁰ represents L⁴-Q.

In various embodiments, Q-L⁴ has the formula:

in which R^(q) is H, substituted or unsubstituted alkyl or substitutedor unsubstituted heteroalkyl and the index e represents an integer from1 to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20.

Also provided is a nucleic acid oligomer, e.g. a probe, prepared using amonomer of the invention and including the components of the monomerfrom which it is synthesized.

Oligomers

Exemplary oligomers include oligonucleotides, oligonucleosides,oligodeoxyribonucleotides (containing 2′-deoxy-D-ribose or modifiedforms thereof), i.e., DNA, oligoribonucleotides (containing D-ribose ormodified forms thereof), i.e., RNA, and any other type of polynucleotidewhich is an N-glycoside or C-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine base. Oligomer as used herein alsoincludes compounds where adjacent nucleomonomers are linked via amidelinkages as previously described (Nielsen et al., Science (1991)254:1497-1500). The enhanced competence of binding by oligomerscontaining the bases of the present invention is believed to beprimarily a function of the base alone. Because of this, elementsordinarily found in oligomers, such as the furanose ring and/or thephosphodiester linkage can be replaced with any suitable functionallyequivalent element. “Oligomer” is thus intended to include any structurethat serves as a scaffold or support for the bases wherein the scaffoldpermits binding to target nucleic acids in a sequence-dependent manner.

Exemplary groups linking nucleomonomers in an oligomer of the inventioninclude (i) phosphodiester and phosphodiester modifications(phosphorothioate, methylphosphonate, etc), (ii) substitute linkagesthat contain a non-phosphorous isostere (formacetal, riboacetal,carbamate, etc), (iii) morpholino residues, carbocyclic residues orother furanose sugars, such as arabinose, or a hexose in place of riboseor deoxyribose and (iv) nucleomonomers linked via amide bonds or acyclicnucleomonomers linked via any suitable substitute linkage.

The oligomers of the invention can be formed using modified andconventional nucleomonomers and synthesized using standard solid phase(or solution phase) oligomer synthesis techniques, which are nowcommercially available. In general, the oligomers can be synthesized bya method comprising the steps of: synthesizing a nucleomonomer oroligomer synthon having a protecting group and a base and a couplinggroup capable of coupling to a nucleomonomer or oligomer; coupling thenucleomonomer or oligomer synthon to an acceptor nucleomonomer or anacceptor oligomer; removing the protecting group; and repeating thecycle as needed until the desired oligomer is synthesized.

The oligomers of the present invention can be of any length includingthose of greater than 40, 50 or 100 nucleomonomers. In variousembodiments, oligomers contain 2-30 nucleomonomers. Lengths of greaterthan or equal to about 8 to 20 nucleomonomers are useful for therapeuticor diagnostic applications. Short oligomers containing 2, 3, 4 or 5nucleomonomers are specifically included in the present invention andare useful, e.g., as synthons.

Oligomers having a randomized sequence and containing fewer than 20,fewer than 15 or fewer than 10 nucleomonomers are useful for primers,e.g., in cloning or amplification protocols that use random sequenceprimers, provided that the oligomer contains residues that can serve asa primer for polymerases or reverse transcriptases.

Oligomers can contain conventional phosphodiester linkages or cancontain phosphodiester modification such as phosphoramidate linkages.These substitute linkages include, but are not limited to, embodimentswherein a moiety of the formula —O—P(O)(S)—O—(“phosphorothioate”),—O—P(S)(S)—O— (“phosphorodithioate”), —O—P(O)— (NR^(o) ₂)—X—,—O—P(O)(R^(o))—O—O—P(S)(R^(o))—O— (“thionoalkylphosphonate”),—P(O)(OR^(p))—X—, —O—C(O)—X—, or —O—C(O)(NR^(p) ₂)—X—, wherein R^(o) isH (or a salt) or alkyl (1-12C) and R^(p) is alkyl (1-9C) and the linkageis joined to adjacent nucleomonomers through an —O— or —S— bonded to acarbon of the nucleomonomer. In various embodiments, the substitutelinkages for use in the oligomers of the present invention includephosphodiester, phosphorothioate, methylphosphonate andthionomethylphosphonate linkages. Phosphorothioate and methylphosphonatelinkages confer added stability to the oligomer in physiologicalenvironments. While not all such linkages in the same oligomer need beidentical, particularly preferred oligomers of the invention containuniformly phosphorothioate linkages or uniformly methylphosphonatelinkages.

Oligomers or the segments thereof are conventionally synthesized, andcan be prepared using a solid support and/or phosphoramidite of theinvention. The synthetic methods known in the art and described hereincan be used to synthesize oligomers containing bases of the invention,as well as other bases known in the art, using appropriately protectednucleomonomers. Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res. (1986)14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides andNucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Lett. (1986)27:5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p 7-24; Reese, C. B. et al., Tetrahedron Lett. (1985)26:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Lett. (1987) 28:3539-3542; Klem, R. E., et al.,International Publication Number WO 92/07864).

As disclosed herein, the invention provides “conjugates” of oligomers.For instance, the oligomers can be covalently linked to variousfunctional components such as, stabilizing moieties (X^(a)),fluorophores, quenchers, intercalators, and substances which interactspecifically with the minor groove of the DNA double helix (minor groovebinders, “MGB”). Other chosen conjugate moieties, can be labels such asradioactive, fluorescent, enzyme, or moieties which facilitate cellassociation using cleavage linkers and the like. Suitable radiolabelsinclude ³²P, ³⁵S, ³H and ¹⁴C; and suitable fluorescent labels includefluorescein, resorufin, rhodamine, BODIPY (Molecular Probes) and texasred; suitable enzymes include alkaline phosphatase and horseradishperoxidase. Additional fluorophores are set forth herein and aregenerally recognized in the art. Other covalently linked moietiesinclude biotin, antibodies or antibody fragments, and proteins, e.g.,transferrin and the HIV Tat protein.

As discussed herein and recognized in the art, the oligomers can bederivatized through any convenient linkage. For example, minor groovebinders, fluorophores, quenchers and intercalators, such as acridine orpsoralen can be linked to the oligomers of the invention through anyavailable —OH or —SH, e.g., at the terminal 5′-position of the oligomer,the 2′-positions of RNA, or an OH, NH₂, COOH or SH incorporated into the5-position of pyrimidines. A derivatized form which contains, forexample, —CH₂CH₂NH₂, —CH₂CH₂CH₂OH or —CH₂CH₂CH₂SH in the 5-position isof use in the present invention. Conjugates including polylysine orlysine can be synthesized as described and can further enhance thebinding affinity of an oligomer to its target nucleic acid sequence(Lemaitre, M. et al., Proc Natl Acad Sci (1987) 84:648-652; Lemaitre, M.et al., Nucleosides and Nucleotides (1987) 6:311-315).

A wide variety of substituents can be attached, including those boundthrough linkages or substitute linkages. The —OH moieties in thephosphodiester linkages of the oligomers can be replaced by phosphategroups, protected by standard protecting groups, or coupling groups toprepare additional linkages to other nucleomonomers, or can be bound tothe conjugated substituent. The 5′-terminal OH can be phosphorylated;the 2′-OH or OH substituents at the 3′-terminus can also bephosphorylated. The hydroxyls can also be derivatized to standardprotecting groups.

Oligomers of the invention can be covalently derivatized to moietiesthat facilitate cell association using cleavable linkers. Linkers usedfor such conjugates can include disulfide linkages that are reducedafter the oligomer-transport agent conjugate has entered a cell.Disulfide-containing linkers of this type have a controllable half life.Such linkers are stable under extracellular conditions relative tointracellular conditions due to the redox potential of the disulfidelinkage.

Donor and Acceptor Moieties

Quenchers

Exemplary solid supports and oligomers of the invention include aquencher covalently attached thereto, optionally through a linker. Invarious embodiments, the quencher is a moiety having a structureaccording to Formula (II)

in which R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. The symbols X, Y and Y′ are members independently selectedfrom reactive functional groups and linkage fragments covalently bindingsaid quencher to L³. The index f is a number selected from 0 to 4 (i.e.,0, 1, 2, 3, or 4), inclusive, such that when (f×s) is greater than 1,the Y′ groups are the same or different. The index m is a numberselected from 0 to 5 (i.e., 0, 1, 2, 3, 4, or 5), inclusive, such thatwhen m is greater than 1, the X groups are the same or different. Theindex n is a number from 0 to 6 (i.e., 0, 1, 2, 3, 4, 5 or 6),inclusive, such that when (n×t) is greater than 1, the Y groups are thesame or different. The index s is a number from 0 to 6 (i.e., 0, 1, 2,3, 4, 5 or 6), inclusive, such that when s is greater than 1 the R³groups are the same or different the index t is a number from 1 to 6(i.e., 1, 2, 3, 4, 5 or 6), inclusive, such that when t is greater than1 the R² groups are the same or different, and when t is 1 and s is 0, amember selected from R¹, R² and combinations thereof is a memberselected from substituted or unsubstituted polycyclic aryl andsubstituted or unsubstituted polycyclic heteroaryl groups.

In various embodiments, the quencher has a structure according toFormula (III):

The solid support and oligomers of the invention also can includequencher according to Formula III in which a member selected from R¹, R²and R³ includes a structure according to Formula IV:

in which R⁴ is a member selected from alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl.

Quenchers of use in various embodiments have a structure according toFormula V:

in which v is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9,10).

In exemplary embodiments, the quencher has a structure according toFormula VI:

in which the symbols R⁵, R⁶ and R⁷ are members independently selectedfrom —NR′R″, substituted or unsubstituted aryl, nitro, substituted orunsubstituted C₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆alkoxy. R′ and R″ are independently selected from H and substituted orunsubstituted C₁-C₆ alkyl. The index n is an integer from 0 to 1. Theindex a is an integer from 0 to 4 (i.e., 0, 1, 2, 3, or 4), such thatwhen a is greater than 1, the R⁵ groups are the same or different. Theindex b is an integer from 0 to 4 (i.e., 0, 1, 2, 3, or 4), such thatwhen (v×b) is greater than 1, the R⁶ groups are the same or different.The index c is an integer from 0 to 5 (i.e., 0, 1, 2, 3, 4, or 5), suchthat when c is greater than 1, the R⁷ groups are the same or different;and v is an integer from 1 to 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10),such that when v is greater than 1, the value of b on each of the bphenyl rings is the same or different.

Various embodiments utilize a quencher having a structure according toFormula VII:

in which R⁵, R⁶ and R⁷ are members independently selected from amine,alkyl amine, substituted or unsubstituted aryl, nitro, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted C₁-C₆ alkoxy.The symbols X¹ and X² represent members independently selected fromC₁-C₆ alkyl or C₁-C₆ substituted alkyl, —OH, —COOH, —NR′R″, —SH,—OP(OX³)(NR′R″) and a linkage fragment covalently binding said quencherto L³. R′ and R″ are members independently selected from the groupconsisting of H, and substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl.

In certain embodiments, the compounds of the invention utilize aquencher having a structure which is a member selected from:

in which X⁵ and X⁶ are members independently selected from H, a reactivefunctional group and a linkage fragment covalently binding said quencher(Q) to L³, with the proviso that at least one of X⁵ and X⁶ is such alinkage fragment.

Thus, the invention provides compounds according to Formula I, includingthe component a component selected from:

As one of skill will appreciate, L³ in the above structures can bereplaced with L⁴ and its attachment to a propyne-modified base.

One of the advantages of the compounds of the invention is that a widerange of energy donor molecules can be used in conjunction with thequencher-functionalized solid supports and oligomers. A vast array offluorophores is known to those of skill in the art. See, for example,Cardullo et al., Proc. Natl. Acad. Sci. USA 85: 8790-8794 (1988);Dexter, D. L., J. of Chemical Physics 21: 836-850 (1953); Hochstrasseret al., Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods inEnzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47: 819-846 (1978); Wanget al, Tetrahedron Letters 31: 6493-6496 (1990); Wang et al., Anal Chem.67: 1197-1203 (1995).

A non-limiting list of exemplary donors that can be used in conjunctionwith the quenchers of the invention is provided in Table 1.

TABLE 1 Suitable moieties that can be selected as donors or acceptors indonor-acceptor energy transfer pairs4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonateN-(4-anilino-1-naphthyl)maleimide anthranilamide BODIPY Brilliant Yellowcoumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyaninedyes cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate quantum dots Reactive Red 4 (Cibacron ™Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine(ROX) 6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 123 rhodamine X isothiocyanatesulforhodamine B sulforhodamine 101 sulfonyl chloride derivative ofsulforhodamine 101 (Texas Red) N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA) tetramethyl rhodamine tetramethyl rhodamine isothiocyanate(TRITC) riboflavin rosolic acid metal chelates, e.g., lanthanidechelates (e.g., europium terbium chelates), ruthenium chelates

There is a great deal of practical guidance available in the literaturefor selecting appropriate donor-acceptor pairs for particular probes, asexemplified by the following references: Pesce et al., Eds.,FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al.,FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York,1970); and the like. The literature also includes references providingexhaustive lists of fluorescent and chromogenic molecules and theirrelevant optical properties for choosing reporter-quencher pairs (see,for example, Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF A ROMATICMOLECULES, 2nd Edition (Academic Press, New York, 1971); Griffiths,COLOUR AND C ONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York,1976); Bishop, Ed., INDICATORS (Pergamon Press, Oxford, 1972); Haugland,HANDBOOK OF FLUORESCENT PROBES AND R ESEARCH CHEMICALS (MolecularProbes, Eugene, 1992) Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE(Interscience Publishers, New York, 1949); and the like. Further, thereis extensive guidance in the literature for derivatizing reporter andquencher molecules for covalent attachment via common reactive groupsthat can be added to a nucleic acid, as exemplified by the followingreferences: Haugland (supra); Ullman et al., U.S. Pat. No. 3,996,345;Khanna et al., U.S. Pat. No. 4,351,760. Thus, it is well within theabilities of those of skill in the art to choose an energy exchange pairfor a particular application and to conjugate the members of this pairto a probe molecule, such as, for example, a nucleic acid, peptide orother polymer.

Generally, it is preferred that an absorbance band of the BHQsubstantially overlap the fluorescence emission band of the donor. Whenthe donor (fluorophore) is a component of a probe that utilizesdonor-acceptor energy transfer, the donor fluorescent moiety and thequencher (acceptor) of the invention are preferably selected so that thedonor and acceptor moieties exhibit donor-acceptor energy transfer whenthe donor moiety is excited. One factor to be considered in choosing thefluorophore-quencher pair is the efficiency of donor-acceptor energytransfer between them. Preferably, the efficiency of FRET between thedonor and acceptor moieties is at least 10%, more preferably at least50% and even more preferably at least 80%. The efficiency of FRET caneasily be empirically tested using the methods both described herein andknown in the art.

The efficiency of energy transfer between the donor-acceptor pair canalso be adjusted by changing the ability of the donor and acceptorgroups to dimerize or closely associate. If the donor and acceptormoieties are known or determined to closely associate, an increase ordecrease in association can be promoted by adjusting the length of alinker moiety, or of the probe itself, between the donor and acceptor.The ability of donor-acceptor pair to associate can be increased ordecreased by tuning the hydrophobic or ionic interactions, or the stericrepulsions in the probe construct. Thus, intramolecular interactionsresponsible for the association of the donor-acceptor pair can beenhanced or attenuated. Thus, for example, the association between thedonor-acceptor pair can be increased by, for example, utilizing a donorbearing an overall negative charge and an acceptor with an overallpositive charge.

In addition to fluorophores that are attached directly to a probe, thefluorophores can also be attached by indirect means. In this embodiment,a ligand molecule (e.g., biotin) is generally covalently bound to theprobe species. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a fluorescent compound, oran enzyme that produces a fluorescent compound by conversion of anon-fluorescent compound. Useful enzymes of interest as labels include,for example, hydrolases, particularly phosphatases, esterases andglycosidases, hydrolases, peptidases or oxidases, particularlyperoxidases, and. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.,as discussed above. For a review of various labeling or signal producingsystems that can be used, see, U.S. Pat. No. 4,391,904.

Presently preferred donors of use in conjunction with BHQ, include, forexample, xanthene dyes, including fluoresceins, cyanine dyes andrhodamine dyes. Many suitable forms of these compounds are widelyavailable commercially with substituents on their phenyl moieties, whichcan be used as the site for bonding or as the bonding functionality forattachment to an nucleic acid. Another group of preferred fluorescentcompounds are the naphthylamines, having an amino group in the alpha orbeta position. Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other donors include3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like.

For clarity of illustration, the discussion below focuses on attachingBHQs and fluorophores to nucleic acids. The focus on nucleic acid probesis not intended to limit the scope of probe molecules to which BHQs canbe attached. Those of skill in the art will appreciate that BHQs canalso be attached to small molecules, proteins, peptides, syntheticpolymers, solid supports and the like using standard syntheticchemistry.

In a presently preferred embodiment, in which the probe is a nucleicacid probe, the reporter molecule is a fluorescein dye (FAM). Thefluorescein moiety is preferably attached to either the 3′- or the5′-terminus of the nucleic acid, although internal sites are alsoaccessible and have utility for selected purposes. Whichever terminusthe FAM derivative is attached to, the BHQ will generally be attached toits antipode, or at a position internal to the nucleic acid chain. TheFAM donor is preferably introduced using a 6-FAM amidite. Differentdonor groups are also preferably introduced using an amidite derivativeof the donor. Alternatively, donor groups comprising reactive functionalgroups (e.g., isothiocyanates, active esters, etc.) can be introducedvia reaction with a reactive functional group on a tether or linker armattached to the nucleic acid (e.g., hexyl amine).

In yet another preferred embodiment, the donor moiety can be attached atthe 3′-terminus of a nucleic acid by the use of a derivatized synthesissupport. For example, TAMRA (tetramethylrhodamine carboxylic acid) isattached to a nucleic acid 3′-terminus using a solid support that isderivatized with an analogue of this fluorophore (BiosearchTechnologies, Inc.)

In view of the well-developed body of literature concerning theconjugation of small molecules to nucleic acids, many other methods ofattaching donor/acceptor pairs to nucleic acids will be apparent tothose of skill in the art. For example, rhodamine and fluorescein dyesare conveniently attached to the 5′-hydroxyl of an nucleic acid at theconclusion of solid phase synthesis by way of dyes derivatized with aphosphoramidite moiety (see, for example, Woo et al., U.S. Pat. No.5,231,191; and Hobbs, Jr., U.S. Pat. No. 4,997,928).

More specifically, there are many linker moieties and methodologies forattaching groups to the 5′- or 3′-termini of nucleic acids, asexemplified by the following references: Eckstein, editor, Nucleic acidsand Analogues: A Practical Approach (IRL Press, Oxford, 1991); Zuckermanet al., Nucleic Acids Research, 15: 5305-5321 (1987) (3′-thiol group onnucleic acid); Sharma et al., Nucleic Acids Research, 19: 3019 (1991)(3′-sulfhydryl); Giusti et al, PCR Methods and Applications, 2: 223-227(1993) and Fung et al., U.S. Pat. No. 4,757,141 (5′-phosphoamino groupvia Aminolink TM II available from P.E. Biosystems, CA.) Stabinsky, U.S.Pat. No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al.,Tetrahedron Letters, 31: 1543-1546 (1990) (attachment viaphosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research,17: 7187-7194 (1989) (3′-amino group), and the like.

Means of detecting fluorescent labels are well known to those of skillin the art. Thus, for example, fluorescent labels can be detected byexciting the fluorophore with the appropriate wavelength of light anddetecting the resulting fluorescence. The fluorescence can be detectedvisually, by means of photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providing theappropriate substrates for the enzyme and detecting the resultingreaction product.

Reactive Functional Groups

The components of the solid supports and oligomers of the invention(e.g., linkers, fluorophore, quenchers, stabilizing moiety are linkedthrough linkage fragments formed by reaction of a first and a secondreactive functional group. The reactive functional groups are ofcomplementary reactivity, and they react to form a covalent link betweentwo components of the oligomers referred to herein as a linkagefragment. With reference to the solid support of Formula I, the reactivefunctional group is found on precursors of L¹, L² and L³, as well as onprecursors of Q, Z^(a) and X^(a). In various examples, X^(a) and L¹ arecovalently joined through a linkage fragment; L² and Z^(a) are joined bya linkage fragment; L³ and Q are joined by a linkage fragment, eachlinkage fragment formed by reaction of reactive functional groups on theprescursors of the named components of the oligomers of the invention.Linkage fragments are present in similar groups of the oligomers ofFormulae VII and VIII.

With respect to the precursors of the components of solid supports,phosphoramidites and oligomers of the invention, reactive functionalgroups can be located at any position on these precursors, e.g., analkyl or heteroalkyl an aryl or heteroaryl nucleus or a substituent onan aryl or heteroaryl nucleus. Similarly, a reactive functional group islocated at any position of an alkyl or heteroalkyl chain. In variousembodiments, when the reactive group is attached to an alkyl (orheteroalkyl), or substituted alkyl (or heteroalkyl) chain, the reactivegroup is preferably located at a terminal position of the chain.

Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive precursors of the oligomers of the invention are thosewhich proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

By way of example, reactive functional groups of use in the presentinvention include, but are not limited to olefins, acetylenes, alcohols,phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids,esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates,amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro,nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones,sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates,sulfenic acids isonitriles, amidines, imides, imidates, nitrones,hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes,ortho esters, sulfites, enamines, ynamines, ureas, pseudoureas,semicarbazides, carbodiimides, carbamates, imines, azides, azocompounds, azoxy compounds, and nitroso compounds. Reactive functionalgroups also include those used to prepare bioconjugates, e.g.,N-hydroxysuccinimide esters, maleimides and the like. Methods to prepareeach of these functional groups are well known in the art and theirapplication to or modification for a particular purpose is within theability of one of skill in the art (see, for example, Sandler and Karo,eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego,1989).

Useful reactive functional group conversions include, for example:

-   -   (a) carboxyl groups which are readily converted to various        derivatives including, but not limited to, active esters (e.g.,        N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,        thioesters, p-nitrophenyl esters), acid halides, acyl        imidazoles, alkyl, alkenyl, alkynyl and aromatic esters;    -   (b) hydroxyl groups, which can be converted to esters, ethers,        halides, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the site of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc;    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds; and    -   (k) phosphoramidites and other standard functional groups useful        in nucleic acid synthesis.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe oligomer of the invention. Alternatively, a reactive functionalgroup can be protected from participating in the reaction by thepresence of a protecting group. Those of skill in the art understand howto protect a particular functional group such that it does not interferewith a chosen set of reaction conditions. For examples of usefulprotecting groups, see, for example, Greene et al., PROTECTIVE GROUPS INORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

Covalent Bonding Moiety

Included in some of the oligomers of the invention is a reactivefunctional group moiety which is capable of effecting at least onecovalent bond between the oligomer and a target sequence. Multiplecovalent bonds can also be formed by providing a multiplicity of suchmoieties. The covalent bond is preferably to a base residue in thetarget strand, but can also be made with other portions of the target,including the sugar or phosphodiester. The reaction nature of the moietywhich effects crosslinker determines the nature of the target in theduplex. Preferred crosslinker moieties include acylating and alkylatingagents, and, in particular, those positioned relative to the sequencespecificity-conferring portion so as to permit reaction with the targetlocation in the strand.

The crosslinker moiety can conveniently be placed as an analogouspyrimidine or purine residue in the sequence of the oligomer. Theplacement can be at the 5′- and/or 3′-ends, the internal portions of thesequence, or combinations of the above. Placement at the termini topermit enhanced flexibility is preferred. Analogous moieties can also beattached to peptide backbones.

Exemplary of alkylating moieties that are useful in the inventioninclude N⁴,N⁴-ethanocytosine and N⁶,N⁶-ethanoadenine.

It is clear that the base need not be a purine or pyrimidine; indeed themoiety to which the reactive function is attached need not be a base atall and may be a sugar, a linker, a quencher, a stabilizing moiety afluorophore or some combination of these components of the oligomers ofthe invention. Any means of attaching the reactive group is satisfactoryso long as the positioning is correct.

Synthesis

Solid supports, monomers (e.g., phosphoramidites) and oligomers of theinvention or the segments thereof are generally conventionallysynthesized. The synthetic methods known in the art and described hereincan be used to synthesize oligomers containing bases of the invention,as well as other bases known in the art, using appropriately protectednucleomonomers. Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res. (1986)14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides andNucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Letters (1986)27:5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p 7-24; Reese, C. B. et al., Tetrahedron Letters (1985)28:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Letters (1987) 28:3539-3542; Klem, R. E., et al.,International Publication Number WO 92/07864).

An exemplary synthesis of a solid support of the invention is set forthin FIG. 1, FIG. 2 and the examples appended hereto.

In an exemplary embodiment, nucleomonomers are directly incorporatedinto oligomers or a convenient fragment thereof using standard synthesisconditions and reagents. Exemplary linkages made by this method includephosphodiester, phosphorothioate, phosphoroamidate, methylphosphonate,phosphorodithioate, carbonate, morpholino carbamate and sulfonate.

In various embodiments, synthesis involves synthesis of short synthons(dimers, trimers, etc.) starting with an appropriate precursor. Thisapproach is suitable for synthesis of linkages includingN-methylhydroxylamine, dimethylhydrazo, sulfamate, carbamate, sulfonate,sulfonamide, formacetal thioformacetal and carbonate.

Oligomers of the invention can be synthesized by any suitable chemistryincluding amidite, triester or hydrogen phosphonate coupling methods andconditions. The oligomers are preferably synthesized from appropriatestarting synthons which are preferably protected at the 5′-position withDMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PACO (phenoxyacetyl), asilyl ether such as TBDMS (t-butyldiphenylsilyl) or TMS (trimethylsilyl)and activated at the 3′-position is an ester, H-phosphonate, an amiditesuch as β-cyanoethylphosphoramidite, a silyl ether such as TBDMS or TMSor t-butyldiphenyl. Alternatively, appropriate uridine or cytidineprecursors such as blocked 5-iodo-2′-deoxyuridine,5-iodo-2′-O-alkyluridine, 5-bromo-2′-deoxyuridine,5-trifluoromethanesulfonate-2′-deoxyuridine, 5-bromo-2′-O-alkyluridineor blocked and protected 5-iodo-2′-deoxycytidine,5-bromo-2′-deoxycytidine, 5-trifluoromethanesulfonate-2′-deoxycytidine,5-iodo-2′-O-alkylcytidine, 5-bromo-2′-O-alkylcytidine can beconveniently incorporated into short oligomers such as dimer, trimer,tetramer, pentamer or longer synthons that are subsequently derivatizedto yield suitable synthons and longer oligomers.

Exemplary synthesis of oligomers containing about 4 or morenucleomonomer residues are accomplished using synthons such as monomers,dimers or trimers that carry a coupling group suitable for use withamidite, H-phosphonate or triester chemistries. The synthon can be usedto link the components of the oligomer via a phosphodiester orphosphorous-containing linkage other than phosphodiester (e.g.,phosphorothioate, methylphosphonate, thionomethylphosphonate,phosphoramidate and the like).

Synthesis of other nonphosphorous-containing substituted linkages can beaccomplished using appropriate precursors as known in the art.

Once the desired nucleic acid is synthesized, it is preferably cleavedfrom the solid support on which it was synthesized and treated, bymethods known in the art, to remove any protecting groups present (e.g.,60° C., 5 h, concentrated ammonia). In those embodiments in which abase-sensitive group is attached to the nucleic acids (e.g., TAMRA), thedeprotection will preferably use milder conditions (e.g.,butylamine:water 1:3, 8 hours, 70° C.). Deprotection under theseconditions is facilitated by the use of quick deprotect amidites (e.g.,dC-acetyl, dG-dmf).

Following cleavage from the support and deprotection, the nucleic acidis purified by any method known in the art, including chromatography,extraction and gel purification. In a preferred embodiment, the nucleicacid is purified using HPLC. The concentration and purity of theisolated nucleic acid is preferably determined by measuring the opticaldensity at 260 nm in a spectrophotometer.

Assays and Oligomeric Probes of the Invention

In various embodiments, the present invention provides an oligomer ofuse in one or more assay formats. In selected embodiments the oligomerparticipates in the generation of a detectable signal upon associationwith or dissociation from its target. The oligomeric probes of theinvention are not limited in use to any particular assay format.Accordingly, the following description is intended to illustrateexemplary assays formats in which the oligomers of the invention finduse, and is not intended to be limiting of the assay formats in whichthe oligomers are of use.

Assays

The following discussion is generally relevant to the assays describedherein. This discussion is intended to illustrate the invention byreference to certain preferred embodiments and should not be interpretedas limiting the scope of probes and assay types in which the compoundsof the invention find use. Other assay formats utilizing the compoundsof the invention will be apparent to those of skill in the art.

In general, to determine the concentration of a target molecule, suchas, for example, a nucleic acid, it is preferable to first obtainreference data in which constant amounts of probe and nucleic acidligand are contacted with varying amounts of target. The fluorescenceemission of each of the reference mixtures is used to derive a graph ortable in which target concentration is compared to fluorescenceemission. For example, a probe that: a) hybridizes to a target-freenucleic acid ligand; and b) has a stem-loop architecture with the 5′ and3′ termini being the sites of fluorescent group and BHQ labeling, can beused to obtain such reference data. Such a probe gives a characteristicemission profile in which the fluorescence emission decreases as thetarget concentration increases in the presence of a constant amount ofprobe and nucleic acid ligand. Then, a test mixture with an unknownamount of target is contacted with the same amount of first nucleic acidligand and second probe, and the fluorescence emission is determined.The value of the fluorescence emission is then compared with thereference data to obtain the concentration of the target in the testmixture.

Multiplex Analyses

In another embodiment, the solid supports and oligomers of the inventionare utilized as a probe or a component of one or more probes used in amultiplex assay for detecting one or more species in a mixture.

Probes based on the solid supports or oligomers of the invention areparticularly useful in performing multiplex-type analyses and assays. Ina typical multiplex analysis, two or more distinct species (or regionsof one or more species) are detected using two or more probes, whereineach of the probes is labeled with a different fluorophore. Preferredspecies used in multiplex analyses relying on donor-acceptor energytransfer meet at least two criteria: the fluorescent species is brightand spectrally well-resolved; and the energy transfer between thefluorescent species and the quencher is efficient.

The solid supports and oligomers of the invention allow for the designof multiplex assays in which more than one quencher structure is used inthe assay. A number of different multiplex assays using the solidsupports or oligomers of the invention will be apparent to one of skillin the art. In one exemplary assay, each of the at least two distinctquenchers is used to quench energy derived from one or more identicalfluorophore. Alternatively, an assay can be practiced in which eachdistinct quencher quenches energy derived from a distinct fluorophore towhich the quencher is “matched.” The fluorophores can be bound to thesame molecule as the quencher or to a different molecule. Moreover,similar to the quencher and the fluorophores, the carrier molecules ofuse in a particular assay system can be the same or different.

In addition to the mixtures described above, the present invention alsoprovides a method for detecting or quantifying a particular molecularspecies. The method includes: (a) contacting the species with a mixturecontaining a solid support or oligomer of the invention; and (b)detecting a change in a fluorescent property of one or more component ofthe resulting mixture, thereby detecting or quantifying the molecularspecies.

The simultaneous use of two or more probes using donor-acceptor energytransfer is known in the art. For example, multiplex assays usingnucleic acid probes with different sequence specificities have beendescribed. Fluorescent probes have been used to determine whether anindividual is homozygous wild-type, homozygous mutant or heterozygousfor a particular mutation. For example, using one quenched-fluoresceinmolecular beacon that recognizes the wild-type sequence and anotherrhodamine-quenched molecular beacon that recognizes a mutant allele, itis possible to genotype individuals for the β-chemokine receptor(Kostrikis et al. Science 279:1228-1229 (1998)). The presence of only afluorescein signal indicates that the individual is wild-type, and thepresence of rhodamine signal only indicates that the individual is ahomozygous mutant. The presence of both rhodamine and fluorescein signalis diagnostic of a heterozygote. Tyagi et al. Nature Biotechnology 16:49-53 (1998)) have described the simultaneous use of four differentlylabeled molecular beacons for allele discrimination, and Lee et al.,BioTechniques 27: 342-349 (1999) have described seven color homogenousdetection of six PCR products.

The quenchers of the present invention can be used in multiplex assaysdesigned to detect and/or quantify substantially any species, including,for example, whole cells, viruses, proteins (e.g., enzymes, antibodies,receptors), glycoproteins, lipoproteins, subcellular particles,organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA, andanalogues thereof), polysaccharides, lipopolysaccharides, lipids, fattyacids, non-biological polymers and small molecules (e.g., toxins, drugs,pesticides, metabolites, hormones, alkaloids, steroids).

Nucleic Acid Probes

The solid supports and oligomers of the invention are usefulnucleic-acid probes and they can be used as components of detectionagents in a variety of DNA amplification/quantification strategiesincluding, for example, 5′-nuclease assay, Strand DisplacementAmplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA),Rolling Circle Amplification (RCA), as well as for direct detection oftargets in solution phase or solid phase (e.g., array) assays.Furthermore, the solid supports and oligomers can be used in probes ofsubstantially any format, including, for example, format selected frommolecular beacons, Scorpion Probes™, Sunrise Probes™, conformationallyassisted probes, light up probes, Invader Detection probes, and TaqMan™probes. See, for example, Cardullo, R., et al, Proc. Natl. Acad. Sci.USA, 85:8790-8794 (1988); Dexter, D. L., J. Chem. Physics, 21:836-850(1953); Hochstrasser, R. A., et al., Biophysical Chemistry, 45:133-141(1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995);Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L., Ann.Rev. Biochem., 47:819-846 (1978); Wang, G., et al., Tetrahedron Letters,31:6493-6496 (1990); Wang, Y., et al., Anal Chem., 67:1197-1203 (1995);Debouck, C., et al., in supplement to nature genetics, 21:48-50 (1999);Rehman, F. N., et al, Nucleic Acids Research, 27:649-655 (1999); Cooper,J. P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, E. M., et al.,Genome Methods, 6:995-1001 (1996); Hochstrasser, R. A., et al.,Biophysical Chemistry, 45:133-141 (1992); Holland, P. M., et al, ProcNatl. Acad. Sci. USA, 88:7276-7289 (1991); Lee, L. G., et al., NucleicAcids Rsch., 21:3761-3766 (1993); Livak, K. J., et al., PCR Methods andApplications, Cold Spring Harbor Press (1995); Vamosi, G., et al,Biophysical Journal, 71:972-994 (1996); Wittwer, C. T., et al.,Biotechniques, 22:176-181 (1997); Wittwer, C. T., et al, Biotechniques,22:130-38 (1997); Giesendorf, B. A. J., et al., Clinical Chemistry,44:482-486 (1998); Kostrikis, L. G., et al., Science, 279:1228-1229(1998); Matsuo, T., Biochemica et Biophysica Acta, 1379:178-184 (1998);Piatek, A. S., et al., Nature Biotechnology, 16:359-363 (1998);Schofield, P., et al., Appl. Environ. Microbiology, 63:1143-1147 (1997);Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., etal., Nature Biotechnology, 14:303-308 (1996); Nazarenko, I. A., et al.,Nucleic Acids Research, 25:2516-2521 (1997); Uehara, H., et al,Biotechniques, 26:552-558 (1999); D. Whitcombe, et al., NatureBiotechnology, 17:804-807 (1999); Lyamichev, V., et al., NatureBiotechnology, 17:292 (1999); Daubendiek, et al., Nature Biotechnology,15:273-277 (1997); Lizardi, P. M., et al., Nature Genetics, 19:225-232(1998); Walker, G., et al., Nucleic Acids Res., 20:1691-1696 (1992);Walker, G. T., et al., Clinical Chemistry, 42:9-13 (1996); and Compton,J., Nature, 350:91-92 (1991).

Thus, the present invention provides a method for detecting a nucleicacid target sequence. The method includes: (a) contacting the targetsequence with a detector nucleic acid (e.g., an oligomer of theinvention); (b) hybridizing the target binding sequence to the targetsequence, thereby altering the conformation of the detector nucleicacid, causing a change in a fluorescence parameter; and (c) detectingthe change in the fluorescence parameter, thereby detecting the nucleicacid target sequence.

In the methods described herein, unless otherwise noted, a preferreddetector nucleic acid includes a single-stranded target bindingsequence. The binding sequence has linked thereto: i) a fluorophore; andii) a quencher; and iii) a stabilizing moiety. Moreover, prior to itshybridization to a complementary sequence, the detector nucleic acid ispreferably in a conformation that allows donor-acceptor energy transferbetween the fluorophore and the quencher when the fluorophore isexcited. Furthermore, in each of the methods described in this section,a change in fluorescence is detected as an indication of the presence ofthe target sequence. The change in fluorescence is preferably detectedin-real time.

Presently preferred nucleic acid probes do not require the nucleic acidto adopt a secondary structure for the probe to function. In thismethod, and unless otherwise noted, the other methods described in thissection, the detector nucleic acid can assume substantially anyintramolecularly associated secondary structure, but this structure ispreferably a member selected from hairpins, stem-loop structures,pseudoknots, triple helices and conformationally assisted structures.Moreover, the intramolecularly base-paired secondary structurepreferably comprises a portion of the target binding sequence.

In another aspect, the invention provides a method for detectingamplification of a target sequence. The method includes the use of anamplification reaction including the following steps: (a) hybridizingthe target sequence and a detector nucleic acid. The detector nucleicacid includes a single-stranded target binding sequence and anintramolecularly associated secondary structure 5′ to the target bindingsequence. At least a portion of the detector sequence forms a singlestranded tail which is available for hybridization to the targetsequence; (b) extending the hybridized detector nucleic acid on thetarget sequence with a polymerase to produce a detector nucleic acidextension product and separating the detector nucleic acid extensionproduct from the target sequence; (c) hybridizing a primer to thedetector nucleic acid extension product and extending the primer withthe polymerase, thereby linearizing the intramolecularly associatedsecondary structure and producing a change in a fluorescence parameter;and (d) detecting the change in the fluorescence parameter, therebydetecting the target sequence.

In yet a further aspect, the invention provides a method of ascertainingwhether a first nucleic acid and a second nucleic acid hybridize. Inthis method, the first nucleic acid is an oligomer (in solution orattached to a solid support) according to the invention. The methodincludes: (a) contacting the first nucleic acid with the second nucleicacid; (b) detecting an alteration in a fluorescent property of a memberselected from the first nucleic acid, the second nucleic acid and acombination thereof, thereby ascertaining whether the hybridizationoccurs.

In various embodiments, the present invention provides probes andmethods of use in detecting polymorphism in nucleic acid targetsequences. Polymorphism refers to the occurrence of two or moregenetically determined alternative sequences or alleles in a population.A polymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at frequencyof greater than 1%, and more preferably greater than 10% or 20% of aselected population. A polymorphic locus may be as small as one basepair. Polymorphic markers include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. The first identified allelic form is arbitrarily designatedas the reference form and other allelic forms are designated asalternative or variant alleles. The allelic form occurring mostfrequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

In an exemplary embodiment, a probe of the invention is utilized todetect a single nucleotide polymorphism. A single nucleotidepolymorphism occurs at a polymorphic site occupied by a singlenucleotide, which is the site of variation between allelic sequences.The site is usually preceded by and followed by highly conservedsequences of the allele (e.g., sequences that vary in less than 1/100 or1/1000 members of the populations). A single nucleotide polymorphismusually arises due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. Singlenucleotide polymorphisms can also arise from a deletion of a nucleotideor an insertion of a nucleotide relative to a reference allele.

A oligomer of the invention bearing both a quencher and a fluorophorecan be used or, alternatively, one or more of the nucleic acids can besingly labeled with a single member of an energy transfer pair (e.g. aquencher or fluorophore). When a nucleic acid singly labeled with aquencher is the probe, the interaction between the first and secondnucleic acids can be detected by observing the interaction between thequencher and the nucleic acid or, more preferably, the quenching by thequencher of the fluorescence of a fluorophore attached to the secondnucleic acid.

In addition to their general utility in probes designed to investigatenucleic acid amplification, polymorphism and detection andquantification, the present solid supports and oligomers can be used insubstantially any nucleic acid probe format now known or laterdiscovered. For example, the solid supports and oligomers of theinvention can be incorporated into probe motifs, such as Taqman™ probes(Held et al., Genome Res. 6: 986-994 (1996), Holland et al., Proc. Nat.Acad. Sci. USA 88: 7276-7280 (1991), Lee et al., Nucleic Acids Res. 21:3761-3766 (1993)), molecular beacons (Tyagi et al., Nature Biotechnology14:303-308 (1996), Jayasena et al., U.S. Pat. No. 5,989,823, issued Nov.23, 1999)) scorpion probes (Whitcomb et al., Nature Biotechnology 17:804-807 (1999)), sunrise probes (Nazarenko et al., Nucleic Acids Res.25: 2516-2521 (1997)), conformationally assisted probes (Cook, R.,copending and commonly assigned U.S. patent Application 2007/0059752,filed Jun. 9, 1999), peptide nucleic acid (PNA)-based light up probes(Kubista et al., WO 97/45539, December 1997), double-strand specific DNAdyes (Higuchi et al., Bio/Technology 10: 413-417 (1992), Wittwer et al.,BioTechniques 22: 130-138 (1997)) and the like. These and other probemotifs with which the present quenchers can be used are reviewed inNONISOTOPIC DNA PROBE TECHNIQUES, Academic Press, Inc. 1992.

The oligomers for use in the probes of the invention can be any suitablesize, and are preferably in the range of from about 10 to about 100nucleotides, more preferably from about 10 to about 80 nucleotides andmore preferably still, from about 20 to about 40 nucleotides. In thedual labeled (fluorophore-quencher) probes, the donor moiety ispreferably separated from the quencher by at least about 6, preferablyat least about 8, preferably at least about 10 nucleotides, and morepreferably by at least about 15 nucleotides. In various embodimentsdonor moiety is preferably attached to either the 3′- or 5′-terminalnucleotides of the probe. The quencher moiety is also preferablyattached to either the 3′- or 5′-terminal nucleotides of the probe. Morepreferably, the donor and acceptor moieties are attached to the 3′- and5′- or 5′- and 3′-terminal nucleotides of the probe, respectively,although internal placement is also useful.

The precise sequence and length of a nucleic acid probe of the inventiondepends in part on the nature of the target polynucleotide to which itbinds. The binding location and length may be varied to achieveappropriate annealing and melting properties for a particularembodiment. Guidance for making such design choices can be found in manyart-recognized references.

In some embodiments, the 3′-terminal nucleotide of the nucleic acidprobe is blocked or rendered incapable of extension by a nucleic acidpolymerase. Such blocking is conveniently carried out by the attachmentof a donor or acceptor moiety to the terminal 3′-position of the nucleicacid probe, either directly or by a linker moiety.

The nucleic acid can comprise DNA, RNA or chimeric mixtures orderivatives or modified versions thereof. Both the probe and targetnucleic acid can be present as a single strand, duplex, triplex, etc.Moreover, the nucleic acid can be modified at the base moiety, sugarmoiety, or phosphate backbone with other groups such as radioactivelabels, minor groove binders, intercalating agents, acetylinicallyunsaturated hydrocarbons, fluoralkyl groups, donor and/or acceptormoieties and the like.

The oligomers of the invention are useful as primers that are discretesequences or as primers with a random sequence. Random sequence primersare generally about 6 or 7 nucleomonomers in length. Such primers can beused in various nucleic acid amplification protocols (PCR, ligase chainreaction, etc) or in cloning protocols. The 5-substitutions of theinvention generally do not interfere with the capacity of the oligomerto function as a primer. Oligomers of the invention having2′-modifications at sites other than the 3′ terminal residue, othermodifications that render the oligomer RNase H incompetent or otherwisenuclease stable can be advantageously used as probes or primers for RNAor DNA sequences in cellular extracts or other solutions that containnucleases. Thus, the oligomers can be used in protocols for amplifyingnucleic acid in a sample by mixing the oligomer with a sample containingtarget nucleic acid, followed by hybridization of the oligomer with thetarget nucleic acid and amplifying the target nucleic acid by PCR, LCRor other suitable methods.

The oligomers derivatized with chelating agents such as EDTA, DTPA oranalogs of 1,2-diaminocyclohexane acetic acid can be utilized in variousin vitro diagnostic assays as described (U.S. Pat. Nos. 4,772,548,4,707,440 and 4,707,352). Alternatively, oligomers of the invention canbe derivatized with crosslinker agents such as5-(3-iodoacetamidoprop-1-yl)-2′-deoxyuridine or5-(3-(4-bromobutyramido)prop-1-yl)-2′-deoxyuridine and used in variousassay methods or kits as described (International Publication No. WO90/14353).

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in subject cells or in recombinant systems, byany suitable method (Graessmann, M., et al., Nucleic Acids Res. (1991)19:53-59).

Conditions that favor hybridization between oligomer of the presentinvention and target nucleic acid molecules can be determinedempirically by those skilled in the art, and can include optimalincubation temperatures, salt concentrations, length and basecompositions of oligonucleotide analogue probes, and concentrations ofoligomer and nucleic acid molecules of the sample. Preferably,hybridization is performed in the presence of at least one millimolarmagnesium and at a pH that is above 6.0. In some embodiments, it may benecessary or desirable to treat a sample to render nucleic acidmolecules in the sample single-stranded prior to hybridization. Examplesof such treatments include, but are not limited to, treatment with base(preferably followed by neutralization), incubation at high temperature,or treatment with nucleases.

In addition, because the salt dependence of hybridization to nucleicacids is largely determined by the charge density of the backbone of ahybridizing oligonucleotide analogue, increasing the ratio of pPNAmonomers in a HypNA-pPNA oligomer or a SerNA-pPNA oligomer of thepresent invention can increase the salt dependence of hybridization.This can be used to advantage in the methods of the present inventionwhere it can in some aspects be desirable to be able to increase thestringency of hybridization by changing salt conditions, for example, orrelease a hybridized nucleic acid by reducing the salt concentration. Inyet other aspects of the present invention, it can be desirable to havehigh-affinity binding of an oligonucleotide analogue of the presentinvention to a nucleic acid in very low salt. In this case, maintaininga ratio of close to 1:1 of HypNA to pPNA monomers in an oligonucleotideanalogue of the present invention is advantageous.

The high degree of specificity of oligomers of the present invention inbinding to target nucleic acid molecules allow the practitioner toselect hybridization conditions that can favor discrimination betweennucleic acid sequences that comprise a stretch of sequence that iscompletely complementary to at least a portion of one or more oligomerand target nucleic acid molecules that comprise a stretch of sequencethat comprises a small number of non-complementary bases within asubstantially complementary sequence. For example, hybridization or washtemperatures can be selected that permit stable hybrids between oligomerof the present invention and target nucleic acid molecules that arecompletely complementary along a stretch of sequence but promotedissociation of hybrids between oligomer of the present invention andtarget nucleic acid molecules that are not completely complementary,including those that comprise one or two base mismatches along a stretchof complementary sequence. The selection of a temperature forhybridization and washes can be dependent, at least in part, on otherconditions, such as the salt concentration, the concentration ofoligomer and target nucleic acid molecules, the relative proportions ofoligomer to target nucleic acid molecules, the length of the oligomersto be hybridized, the base composition of the oligomer and targetnucleic acid molecules, the monomer composition of the oligonucleotideanalogue molecules, etc. In addition, when selecting for conditions thatfavor stable hybrids of completely complementary molecules and disfavorstable hybrids between oligomer and target nucleic acid molecules thatare mismatched by one or more bases, additional conditions can be takeninto account, and, where desirable, altered, including but not limitedto, the length of the oligonucleotide analogue to be hybridized, thelength of the stretch of sequence of complementarity between oligomerand target nucleic acid molecules, the number of non-complementary baseswithin a stretch of sequence of complementarity, the identity ofmismatched bases, the identity of bases in the vicinity of themismatched bases, and the relative position of any mismatched basesalong a stretch of complementarity. (See, for example, Examples 20, 27,28, and 29.) Those skilled in the art of nucleic acid hybridizationwould be able to determine favorable hybridization and wash conditionsin using oligomer of the present invention for hybridization to targetnucleic acid molecules, depending on the particular application.“Favorable conditions” can be those favoring stable hybrids betweenoligomer and target nucleic acid molecules that are, at least in part,substantially complementary, including those that comprise one or moremismatches.

“Favorable conditions” can be those favoring stable hybrids betweenoligomer and target nucleic acid molecules that are, at least in part,completely complementary and disfavor or destabilized hybrids betweenmolecules that are not completely complementary.

Using methods such as those disclosed herein, the melting temperature ofoligomer of the present invention hybridized to target nucleic acidmolecules of different sequences can be determined and can be used indetermining favorable conditions for a given application. It is alsopossible to empirically determine favorable hybridization conditions by,for example, hybridizing target nucleic acid molecules to oligomer thatare attached to a solid support and detecting hybridized complexes.

Target nucleic acid molecules that are bound to solid supports oroligomeric probes of the present invention can be conveniently andefficiently separated from unbound nucleic acid molecules of the surveypopulation by the direct or indirect attachment of oligomer probes to asolid support. A solid support can be washed at high stringency toremove nucleic acid molecules that are not bound to oligomer probes.However, the attachment of oligomer probes to a solid support is not arequirement of the present invention. For example, in some applicationsbound and unbound nucleic acid molecules can be separated bycentrifugation through a matrix or by phase separation or some by otherforms of separation (for example, differential precipitation) that canoptionally be aided by chemical groups incorporated into the oligomerprobes (see, for example, U.S. Pat. No. 6,060,242 issued May 9, 2000, toNie et al.).

Nucleic Acid Capture Probes

In one embodiment, an immobilized nucleic acid comprising a quencher anda stabilizing moiety is used as a capture probe. The nucleic acid probecan be attached directly to a solid support, for example by attachmentof the 3′- or 5′-terminal nucleotide of the probe to the solid support.More preferably, however, the probe is attached to the solid support bya linker (supra). The linker serves to distance the probe from the solidsupport. The linker is most preferably from about 5 to about 30 atoms inlength, more preferably from about 10 to about 50 atoms in length.

In various embodiments, the solid support is also used as the synthesissupport in preparing the oligomer (probe). The length and chemicalstability of the linker between the solid support and the first 3′-unitof nucleic acid play an important role in efficient synthesis andhybridization of support bound nucleic acids. The linker arm ispreferably sufficiently long so that a high yield (>97%) can be achievedduring automated synthesis. The required length of the linker willdepend on the particular solid support used. For example, a six atomlinker is generally sufficient to achieve a >97% yield during automatedsynthesis of nucleic acids when high cross-linked polystyrene is used asthe solid support. The linker arm is preferably at least 20 atoms longin order to attain a high yield (>97%) during automated synthesis whenCPG is used as the solid support.

Hybridization of a probe immobilized on a solid support generallyrequires that the probe be separated from the solid support by at least30 atoms, more preferably at least 50 atoms. In order to achieve thisseparation, the linker generally includes a spacer positioned betweenthe linker and the 3′-terminus. For nucleic acid synthesis, the linkerarm is usually attached to the 3′-OH of the 3′-terminus by an esterlinkage which can be cleaved with basic reagents to free the nucleicacid from the solid support.

A wide variety of linkers are known in the art, which may be used toattach the nucleic acid probe to the solid support. The linker may beformed of any compound, which does not significantly interfere with thehybridization of the target sequence to the probe attached to the solidsupport. The linker may be formed of, for example, a homopolymericnucleic acid, which can be readily added on to the linker by automatedsynthesis. Alternatively, polymers such as functionalized polyethyleneglycol can be used as the linker. Such polymers are presently preferredover homopolymeric nucleic acids because they do not significantlyinterfere with the hybridization of probe to the target nucleic acid.Polyethylene glycol is particularly preferred because it is commerciallyavailable, soluble in both organic and aqueous media, easy tofunctionalize, and completely stable under nucleic acid synthesis andpost-synthesis conditions.

The linkage fragments between the solid support, the linker and theprobe are preferably not cleaved during synthesis or removal of baseprotecting groups under basic conditions at high temperature. Theselinkages can, however, be selected from groups that are cleavable undera variety of conditions. Examples of presently preferred linkagesinclude carbamate, ester and amide linkages.

Detection of Nucleic Acids in Samples

Solid supports and oligomers of the present invention can be used fordetection of nucleic acids. Such detection methods include: providing asample, contacting at least one oligonucleotide analogue of the presentinvention with the sample under conditions that allow hybridization ofoligomer to nucleic acid molecules, and detecting one or more nucleicacid molecules of the sample that have hybridized to one or moreoligomer of the present invention.

A sample can be from any source, and can be a biological sample, such asa sample from an organism or a group of organisms from the same ordifferent species. A biological sample can be a sample of bodily fluid,for example, a blood sample, serum sample, lymph sample, a bone marrowsample, ascites fluid, pleural fluid, pelvic wash fluid, ocular fluid,urine, semen, sputum, or saliva. A biological sample can also be anextract from cutaneous, nasal, throat, or genital swabs, or extracts offecal material. Biological samples can also be samples of organs ortissues, including tumors. Biological samples can also be samples ofcell cultures, including both cell lines and primary cultures of bothprokaryotic and eukaryotic cells.

A sample can be from the environment, such as from a body of water orfrom the soil, or from a food, beverage, or water source, an industrialsource, workplace area, public area, or living area. A sample can be anextract, for example a liquid extract of a soil or food sample. A samplecan be a solution made from washing or soaking, or suspending a swabfrom, articles such as tools, articles of clothing, artifacts, or othermaterials.

A sample can be an unprocessed or a processed sample; processing caninvolve steps that increase the purity, concentration, or accessibilityof components of the sample to facilitate the analysis of the sample. Asnonlimiting examples, processing can include steps that reduce thevolume of a sample, remove or separate components of a sample,solubilize a sample or one or more sample components, or disrupt,modify, expose, release, or isolate components of a sample. Nonlimitingexamples of such procedures are centrifugation, precipitation,filtration, homogenization, cell lysis, binding of antibodies, cellseparation, etc. For example, in some preferred embodiments of thepresent invention, the sample is a blood sample that is at leastpartially processed, for example, by the removal of red blood cells, byconcentration, by selection of one or more cell or virus types (forexample, white blood cells or pathogenic cells), or by lysis of cells,etc.

Exemplary samples include a solution of at least partially purifiednucleic acid molecules. The nucleic acid molecules can be from a singlesource or multiple sources, and can comprise DNA, RNA, or both. Forexample, a solution of nucleic acid molecules can be a sample that wassubjected to any of the steps of cell lysis, concentration, extraction,precipitation, nucleic acid selection (such as, for example, poly A RNAselection or selection of DNA sequences comprising Alu elements), ortreatment with one or more enzymes. The sample can also be a solutionthat comprises synthetic nucleic acid molecules.

An oligomer or solid support of the present invention can be anyoligomer format disclosed herein, or any oligomer comprising a monomer,dimer or non nucleic acid component (e.g., linker, fluorophore,quencher, stabilizing moiety) disclosed herein. An oligonucleotideanalogue used in the methods of the present invention can be of anylength and of any base composition, and can comprise one or more nucleicacid moieties, peptides, proteins lipids, carbohydrates, steroids, andother biochemical and chemical moieties. An oligonucleotide analogue ofthe present invention can be provided in solution or bound to a solidsupport. In some preferred embodiments of the present invention, theoligomer comprise HypNA and pPNA residues, and can comprise HypNA andpPNA residues in ratios from about 2:1 to about 1:3. More preferably,the oligomer used in the methods of the present invention compriseratios of HypNA to pPNA residues from about 1:1 to about 1:2.

Detection methods for bound nucleic acids are well known in the art, andcan include the use of a detectable label that is attached to orincorporated into nucleic acid molecules of the survey population orthat becomes bound to or incorporated into a hybridized target nucleicacid molecule or hybridized target nucleic acid molecule complex.Detectable labels for nucleic acid molecules are well-known in the art,and comprise fluorescent molecules such as fluorophores (including thoseset forth herein), radioisotopes, mass-altered chemical groups, specificbinding members such as biotin that can be detected by signal-generatingmolecules, and the like. Detectable labels can also be incorporated intoor attached to oligomer of the present invention, for example, in caseswhere sandwich hybridization using a signal oligomer is used fordetection, or detection is performed using a specific binding membersuch as an antibody that recognizes oligomer/target nucleic acidmolecule complexes. Solid supports can be scanned, exposed to film,visually inspected, etc. to determine the presence of a detectable labeland thereby determine the binding of a target nucleic acid molecule toan oligomer immobilized on a solid support such as those of theinvention.

Kits

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of syntheses using the solid supports of theinvention and assays using oligomers of the invention, as describedabove. The kits of the invention typically comprise a solid support oroligomer of the invention, either present as a chemically reactivespecies useful for preparing conjugates, or present as a completedoligomer where the oligomer is a specific binding pair member. The kitoptionally further comprises one or more buffering agents, typicallypresent as an aqueous solution. The kits of the invention optionallyfurther comprise additional detection reagents, a purification mediumfor purifying the resulting labeled substance, luminescence standards,enzymes, enzyme inhibitors, organic solvent, or instructions forcarrying out an assay of the invention. Other formats for kits will beapparent to those of skill in the art and are within the scope of thepresent invention.

By way of summary, the in exemplary embodiments, the present inventionprovides: A compound having a structure according to Formula I:

wherein X^(a) is a stabilizing moiety which is a member selected fromfluoroalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. L¹, L², L³ and L⁴ are linkers independentlyselected from substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl. Y^(a) is a member selected from CR^(a), andN, wherein R^(a) is a member selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Z^(a)is a member selected from a solid support, OR^(b) and NR^(b)R^(b′),wherein R^(b) and R^(b′) are independently selected from H, substitutedor unsubstituted alkyl and substituted or unsubstituted heteroalkyl. Qis a quencher of fluorescent energy comprising a member selected from:

-   -   (a) at least three residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl wherein a first said residue is        covalently linked to a second said residue via a first exocyclic        diazo bond and a member selected from said first residue and        said second residue is covalently linked to the third residue        through a second diazo bond; and    -   (b) at least two residues, each independently selected from        substituted or unsubstituted aryl, and substituted or        unsubstituted heteroaryl wherein at least two of said residues        are covalently linked via an exocyclic diazo bond, with the        proviso that at least one said residue is a member selected from        substituted or unsubstituted polycyclic aryl and substituted or        unsubstituted polycyclic heteroaryl groups;        R^(c) is a member selected from H, substituted or unsubstituted        alkyl, substituted or unsubstituted heteroalkyl, substituted or        unsubstituted aryl, substituted or unsubstituted heteroaryl and        a phosphorus-containing linker covalently bound to a nucleic        acid.

A compound according to the preceding paragraph wherein R^(b) isfluoroalkyl and R^(c) is a phosphorus-containing linker covalently boundto a nucleic acid.

A compound according to any preceding paragraph wherein said quencherhas a structure according to Formula (II)

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; X, Y and Y′ are members independently selected from areactive functional group and a linkage fragment covalently binding saidquencher to L³ is with the proviso that at least one of X, Y and Y′ issaid linkage fragment; f is an integer from 0 to 4, such that when (f×s)is greater than 1, the Y′ groups are the same or different; m is aninteger from 0 to 5, such that when m is greater than 1, the X groupsare the same or different; n is an integer from 0 to 6, such that when(n×t) is greater than 1, the Y groups are the same or different; s is aninteger from 0 to 6, such that when s is greater than 1 the R³ groupsare the same or different; and t is an integer from 1 to 6, such thatwhen t is greater than 1 the R² groups are the same or different, andwhen t is 1 and s is 0, a member selected from R¹, R² and combinationsthereof is a member selected from substituted or unsubstitutedpolycyclic aryl and substituted or unsubstituted polycyclic heteroarylgroups.

A compound according to any of the preceding paragraphs, wherein saidquencher has a structure according to Formula (III):

A compound according to any of the preceding paragraphs, wherein amember selected from R¹, R² and R³ includes a structure according toFormula IV:

wherein R⁴ is a member selected from alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl.

A compound according to any preceding paragraph, wherein said quencherhas a structure according to Formula V:

wherein v is an integer from 1 to 10.

A compound according to any preceding paragraph, wherein said quencherhas a structure according to Formula VI:

wherein R⁵, R⁶ and R⁷ are members independently selected from —NR′R″,substituted or unsubstituted aryl, nitro, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆ alkoxy. R′ and R″are independently selected from H and substituted or unsubstituted C₁-C₆alkyl. The index n is an integer from 0 to 1. The index a is an integerfrom 0 to 4, such that when a is greater than 1, the R⁵ groups are thesame or different. The index b is an integer from 0 to 4, such that when(v×b) is greater than 1, the R⁶ groups are the same or different. Theindex c is an integer from 0 to 5, such that when c is greater than 1,the R⁷ groups are the same or different; and the index v is an integerfrom 1 to 10, such that when v is greater than 1, the value of b on eachof the b phenyl rings is the same or different.

A compound according to any previous paragraph, wherein said quencherhas a structure according to Formula VI:

wherein X¹ and X² are members independently selected from C₁-C₆ alkyl orC₁-C₆ substituted alkyl, —OH, —COOH, —NR′R″, —SH, —OP(OX³)(NR^(g)R^(h))and a linkage fragment covalently binding said quencher to L³, with theproviso that at least one of R⁵, R⁶, X¹ and X² comprises said linkagefragment, wherein R^(g) and R^(h) are members independently selectedfrom the group consisting of H, and substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl.

A compound according to any preceding paragraph, wherein said quencherhas a structure which is a member selected from:

wherein X⁵ and X⁶ are members independently selected from H, a reactivefunctional group and a linkage fragment covalently binding said quencherto L³, with the proviso that at least one of X⁵ and X⁶ is said linkagefragment.

A compound according any preceding paragraph, having a structureaccording to Formula VII:

X^(b) is a member selected from O and S. X^(c) is a member selected fromOR⁸, SR⁸ and NR⁸ R^(8a). R⁸ and R^(8a) are members independentlyselected from H, and substituted or unsubstituted alkyl, or OR⁸ and SR⁸are selected from O⁻M⁺ and S⁻M⁺, respectively. M+ is a metal ion. R⁹ isa member selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl and substituted or unsubstituted heteroaryl and a nucleic acidoptionally connected through a phosphorus-containing linker.

A compound according to any preceding paragraph, having a structureaccording to Formula VIII:

wherein Q¹ is a fragment of said quencher, said fragment comprising amember selected from:

-   -   (c) two moieties selected from substituted or unsubstituted aryl        and substituted or unsubstituted heteroaryl, said two moieties        being linked through an exocyclic diazo bond; and    -   (d) a moiety selected from substituted or unsubstituted        polycyclic aryl and substituted or unsubstituted polycyclic        heteroaryl groups; and each R¹² is a member selected from the        group of aryl substituents.

A compound according to any preceding paragraph, wherein Z^(a) is asolid support and L²-Z^(a) comprises a structure according to FormulaIX:

SS is said solid support.

A compound according to any preceding paragraph, wherein X^(a) isselected from intercalating agents and minor groove binders.

A compound according to any preceding paragraph, wherein said nucleicacid comprises a base having a formula selected from:

wherein R¹⁰ is a member selected from an alkynyl and a fluoroalkylmoiety.

A nucleic acid comprising at least one base, having a formula selectedfrom:

wherein R¹⁰ is a member selected from an alkynyl and a fluoroalkylmoiety; and a quencher of fluorescence energy comprising at least threeresidues, each independently selected from substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and combinations thereof,wherein at least two of said residues are covalently linked via anexocyclic diazo bond.

The materials and methods of the present invention are furtherillustrated by the examples which follow. These examples are offered toillustrate, but not to limit the claimed invention.

EXAMPLES Example 1 Synthesis of BHQ1-Plus CPG

1-O-DMT-2-(N-Fmoc-4-aminobutyryl-1,3-propane diol 1 (FIG. 1) wasprepared according to Nelson, et al., U.S. Patent No. 5,942,610 (1999),except that a mixture of THF, water and Na₂CO₃ was used (instead of DMF)during the Fmoc addition step leading to 1. After purification by columnchromatography, the Fmoc group of 1 was removed with methylamine inethanol, and after rigorous removal of the methylamine by co-evaporationwith pyridine, BOP activated 9-acridinecarboxylic acid was added in DMF.The resulting 1-O-DMT-2 -(N-carboxyacridine-4-aminobutyryl)-1,3-propanediol, 2, was isolated by removal of solvents and column chromatography.After further drying by co-evaporation from dry pyridine, diglycolicacid anhydride was added to produce1-O-DMT-2-(N-carboxyacridine-4-aminobutyryl)-3-O-diglycolate-1,3-propane diol, 3, isolated as its triethylamine saltafter column chromatography. 3 was added to aminopropyl CPG, afteractivation with BOP and NMM, to give DMT acridine CPG 4. Unreacted aminegroups on the CPG were capped (acetylated) with a mixture of aceticanhydride and n-methylimidazole in acetonitrile. The DMT loading of CPG4was determined to be from 45 to 80 micromles per gram, found bydetritylation (3% DCA / DCM) of a quantity of the dried support andcolorometric analysis.

According to FIG. 2, CPG 4 was detritylated (3% DCA/DCM), washed anddried by co-evaporation with pyridine (Scheme 2). BHQ1 DMT amidite(Cook, et al. U.S. Pat. No. 7,109,312 (2006)) was added to thedetritylated CPG and activated with 0.5 M ethylthiotetrazole. After 2minutes, the CPG was washed with MeCN and oxidized with a solution ofiodine, pyridine and water in THF. Support 5 was washed, acetylated asabove, then well washed and dried. The final DMT loading was 30-50micromoles/g.

Example 2 Synthesis of 5′-DMTdU-5-Alkynyl(BHQ1)3′-diisopropyl cyanoethylphosphoramidite

Starting with 5-iodouridine 6 the compound 9 was made in a 5 stepsynthesis. First, Sonogashira coupling (JACS 2005, 127, 15071) withpropargylamine trifluoroacetamide, copper iodide andtetrakistriphenylphosphine palladium (0) in DMF with triethylamine gave5-propargyl(trifluoroacetamide) nucleotide 7 in 37% yield after silicachromatography. The nucleoside was dried well and the 5′DMT group wasadded by using DMT chloride in dry pyridine to give 8 in 87% yield afterchromatography. The TFA amine protecting group was removed withmethylamine in ethanol, to give 5-propargylamine nucleoside. Next, BHQ1C3 carboxylic acid was added to the amine nucleoside with BOP andN-methylmorpholine to produce 5′-DMTdU-5-alkynyl(BHQ1) nucleoside 11.

After drying by evaporation from pyridine, the nucleoside 11 wasconverted into 5′-DMTdU-5 -alkynyl(BHQ1)3′-diisopropyl cyanoethylphosphoramidite 12 with tetraisopropyl cyanoethyl phosphoramidite andtetrazole in a mixture of dry acetonitrile and dichloromethane. Afterpurification on a silica column with a gradient of methanol indichloromethane with 2% pyridine, 700 mg of 12 were obtained. Thephosphramidite was coupled to 5′-TTTTTTTTTT-3′(SEQ ID NO:1) immobilizedon CPG with standard phosphoramidite chemistry. Analysis of the productby ESMS showed a mass of 3809. 5 AMU (Calc'd 3808.5).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto included within the spirit and purview of this application and areconsidered within the scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A compound of a structure according to Formula I:

wherein X^(a) is a stabilizing moiety which is a member selected fromfluoroalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl; L^(l), L², and L³ are linkers independentlyselected from substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl; Y^(a) is a member selected from CR^(a), and Nwherein R^(a) is a member selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl; Z^(a) is a memberselected from a solid support, OR^(b) and NR^(b)R^(b′) wherein R^(b) andR^(b′)are independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl; Q is a quencher offluorescent energy comprising a member selected from: (a) at least threeresidues, each independently selected from substituted or unsubstitutedaryl, and substituted or unsubstituted heteroaryl wherein a first saidresidue is covalently linked to a second said residue via a firstexocyclic diazo bond and a member selected from said first residue andsaid second residue is covalently linked to the third residue through asecond diazo bond; and (b) at least two residues, each independentlyselected from substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl wherein at least two of said residues arecovalently linked via an exocyclic diazo bond, with the proviso that atleast one said residue is a member selected from substituted orunsubstituted polycyclic aryl and substituted or unsubstitutedpolycyclic heteroaryl groups; R^(c) is a member selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl and a phosphorus-containing linker covalentlybound to a nucleic acid.
 2. The compound according to claim 1, whereinR^(b) is fluoroalkyl and R^(c) is a phosphorus-containing linkercovalently bound to a nucleic acid.
 3. The compound according to claim1, wherein said quencher is of a structure according to Formula (II)

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; X, Y and Y′ are members independently selected from areactive functional group and a linkage fragment covalently binding saidquencher to L³ is with the proviso that at least one of X, Y and Y′ issaid linkage fragment; f is an integer from 0 to 4, such that when (f×s)is greater than 1, the Y′ groups are the same or different; m is aninteger from 0 to 5, such that when m is greater than 1, the X groupsare the same or different; n is an integer from 0 to 6, such that when(n×t) is greater than 1, the Y groups are the same or different; s is aninteger from 0 to 6, such that when s is greater than 1 the R³ groupsare the same or different; and t is an integer from 1 to 6, such thatwhen t is greater than 1 the R² groups are the same or different, andwhen t is 1 and s is 0, a member selected from R¹, R² and combinationsthereof is a member selected from substituted or unsubstitutedpolycyclic aryl and substituted or unsubstituted polycyclic heteroarylgroups.
 4. The compound according to claim 3, wherein said quencher isof a structure according to Formula (III):


5. The compound according to claim 3, wherein a member selected from R¹,R² and R³ includes a structure according to Formula IV:

wherein R⁴ is a member selected from alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl and substituted heteroaryl.
 6. The compoundaccording to claim 1, wherein said quencher is of a structure accordingto Formula V:

wherein R¹, R² and R³ are members independently selected fromsubstituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; X and Y are members independently selected from a reactivefunctional group and a linkage fragment covalently binding said quencherto L³, with the proviso that at least one of X and Y is said linkagefragment; m is an integer from 0 to 5, such that when m is greater than1, the X groups are the same or different; and v is an integer from 1 to10.
 7. The compound according to claim 1, wherein said quencher is of astructure according to Formula VI:

wherein R⁵, R⁶ and R⁷ are members independently selected from —NR′R″,substituted or unsubstituted aryl, nitro, substituted or unsubstitutedC₁-C₆ alkyl, and substituted or unsubstituted C₁-C₆ alkoxy, wherein R′and R″ are independently selected from H and substituted orunsubstituted C₁-C₆ alkyl; X and Y are members independently selectedfrom a reactive functional group and a linkage fragment covalentlybinding said quencher to L³, with the proviso that at least one of X andY is said linkage fragment; n is an integer from 0 to 1; a is an integerfrom 0 to 4, such that when a is greater than 1, the R⁵ groups are thesame or different; b is an integer from 0 to 4, such that when (v×b) isgreater than 1, the R⁶ groups are the same or different; c is an integerfrom 0 to5, such that when c is greater than 1, the R⁷ groups are thesame or different; and v is an integer from 1 to 10, such that when v isgreater than 1, the value of b on each of the v phenyl rings is the sameor different.
 8. The compound according to claim 7, wherein saidquencher is of a structure according to the formula:

wherein X¹ and X² are members independently selected from C₁-C₆ alkyl orC₁-C₆ substituted alkyl, and a linkage fragment covalently binding saidquencher to L³, with the proviso that at least one of R⁵, R⁶, X¹ and X²comprises said linkage fragment.
 9. The compound according to claim 1,wherein said quencher is of a structure which is a member selected from:

wherein X⁵ and X⁶ are members independently selected from H, a reactivefunctional group and a linkage fragment covalently binding said quencherto L³, with the proviso that at least one of X⁵ and X⁶ is said linkagefragment.
 10. The compound according to claim 1, wherein said compoundis of a structure according to Formula VII:

wherein v, t and s are independently selected from the integers from 1to 10; X^(b) is a member selected from O and S; X^(c) is a memberselected from OR⁸, SR⁸ and NR⁸ R^(8a) wherein R⁸ and R^(8a) are membersindependently selected from H, and substituted or unsubstituted alkyl,or OR⁸ and SR⁸ are selected from O⁻M⁺ and S⁻M⁺, respectively whereinM+is a metal ion; R⁹ is a member selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl and a nucleic acid connected through a phosphorus-containinglinker.
 11. The compound according to claim 10, wherein said compound isof a structure according to Formula VIII:

wherein Q¹ is a fragment of said quencher, said fragment comprising amember selected from: (a) two moieties selected from substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl, said twomoieties being linked through an exocyclic diazo bond; and (b) a moietyselected from substituted or unsubstituted polycyclic aryl andsubstituted or unsubstituted polycyclic heteroaryl groups; and each R¹²is a member selected from the group of aryl substituents.
 12. Thecompound according to claim 1, wherein Z^(a) is a solid support andL²-Z^(a) comprises a structure according to Formula IX:

wherein u and y are independently selected from the integers from 1 to10; and SS is said solid support.
 13. The compound according to claim 1,wherein said nucleic acid comprises a base of a formula selected from:

wherein R¹⁰ is a member selected from an alkynyl and a fluoroalkylmoiety.
 14. A nucleic acid comprising at least one base of a formulaselected from:

wherein R¹⁰ is a member selected from an alkynyl and a fluoroalkylmoiety; and a quencher of fluorescence energy comprising at least threeresidues, each independently selected from substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl and combinations thereof,wherein at least two of said residues are covalently linked via anexocyclic diazo bond.
 15. The compound according to claim 1, whereinX^(a) is a member selected from intercalating agents and minor groovebinders.
 16. The compound according to claim 15, wherein saidintercalating agent is a member selected from acridines, anthracenes,anthracyclines, anthracyclinone, methylene blue, indole, anthraquinone,quinoline, isoquinoline, dihydroquinones, tetracyclines, psoralens,coumarins, ethidium halides, ethidium homodimers, homodimeric oxazoleyellow (YOYO), thiazole orange (TOTO), dynemicins,1,10-phenanthroline-copper, calcheamicin, porphyrins, distamycins,netropcins, and viologens.
 17. The compound according to claim 16,wherein said intercalating agent is acridine.
 18. The compound accordingto claim 1, wherein said nucleic acid is a probe.
 19. The compoundaccording to claim 18, wherein said probe is selected from molecularbeacons, scorpion probes, sunrise probes, conformationally assistedprobes, light up probes, invader detection probes, and taqman probes.20. The compound according to claim 1, wherein Z^(a) is a solid support.21. The compound according to claim 1, wherein said compound is of thestructure:

wherein DMT is dimethoxytrityl.